Method and a system for determination of particles in a liquid sample

ABSTRACT

The present invention relates to a method for the assessment of quantity and quality parameters of biological particles in a liquid analyte material. The method comprises applying a volume of a liquid sample to an exposing domain from which exposing domain electromagnetic signals from the sample in the domain can pass to the exterior, and exposing, onto an array of active detection elements such as CCD-elements, a spatial representation of electromagnetic signals having passed from the domain, the representation being detectable as an intensity by individual active detection elements, under conditions permitting processing of the intensities detected by the array of detection elements during the exposure in such a manner that representations of electromagnetic signals from the biological particles are identified as distinct from representations of electromagnetic signals from background signals. The size of the volume of the liquid sample is sufficiently large to permit the assessment of the quantity and quality parameters to fulfill a predetermined requirement to the statistical quality of the assessment based on substantially one exposure.

This application is a Continuation of application Ser. No. 11/130,717filed May 17, 2005, which is a Continuation of application Ser. No.10/727,832 filed Dec. 4, 2003, now issued as U.S. Pat. No. 6,919,960,which is a Continuation of application Ser. No. 09/403,958, filed Nov.1, 1999, now issued as U.S. Pat. No. 6,710,879, which is a NationalStage of Application PCT/DK98/00175 filed May 5, 1998 which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to a method and a system for the determination orassessment of at least one quantity parameter and/or at least onequality parameter of biological particles in a liquid analyte material.As an important quantity parameter can be mentioned the number ofbiological particles in a volume of the analyte material, such as, e.g.,the number of somatic cells in milk or blood, or the number of bacteriain a urine sample. Another important example of a quantity parameterwhether or not an analyte, such as a liquid analyte derived by selectiveenrichment of a food sample, contains a particular bacterial species,such as Salmonella typhimurium. As examples of quality parameters may bementioned morphological properties of biological particles such as sizeand/or shape, or identification of one or more types of biologicalparticles in a mixture of more than one types of biological particles.

DESCRIPTION OF RELATED ART

Determinations or assessments of the above types have been performed byvarious methods. One of these methods is flow cytometry; instrument forperforming flow cytometry are available, e.g., from Becton, Dickinsonand Company, Franklin Lakes. Flow cytometry requires rather elaborateand high cost equipment, partly because of the high accuracy of flowrate necessary to give reliable results, and partly because the highsensitivity needed to detect the weak signals from the particles inquestion during the relative short period of time the particle ispresent in the detector.

Another known method for the determination of somatic cells or bacteriain milk is based on the detection of signals from particles which aredispersed on the rim of a polished rotating disc, one such instrumentavailable from Foss Electric, Hillerød. The accuracy in the assessmentof the number of particles using this method is dependent on thephysical shape of the thin film of sample dispersed on the disk, andhigh sensitivity is needed to detect the weak signals from the particlesin question in the course of the relative short period of time theparticle is present in the detector.

One known method for the determination of somatic cells in milk based onspreading a film of milk onto a ribbon-like film which is then analysedby the means of a microscope, cf. European patent 0 683 395. This methodappears to requires a complex mechanical solution in order to workreliably.

Due to the relative high complexity and cost the instruments used today,most of the assessments of biological particles are carried out on in alaboratory where skilled operators operate the instruments.

DESCRIPTION OF THE INVENTION

The present invention offers substantial simplification of theassessment of quantity parameters and/or quality parameters ofbiological particles in liquid analyte materials and therefore makes itpossible for operators without any particular skill in this fields oftechnique to perform the assessment. In particular, the invention makesit possible to perform the assessment in the clinic or on the farm wherethe sample is taken, thus making the results of the assessment availablefor the user substantially immediately after the sample material hasbeen collected.

The physical dimension of an instrument based on the present inventionis also such that the instrument will be well suited for transport, thusmaking it possible for medical doctors or veterinarians to transport theinstrument to or on a location where the analysis is needed. Theprinciple of measurement of the present invention provides a majorimprovement in the assessment of biological particles, such as DNAcontaining particles, e.g. somatic cells or bacteria, or red bloodcells, in a liquid analyte material, such as milk, blood or urine,compared to the methods hitherto used for this purpose.

This invention extends the capabilities of prior devices and methods toenable more simple and reliable assessment of biological particles inliquid analyte material. The properties which can be assessed are thenumber of particles in a volume of the analyte material, anymorphological properties such as size or area of the particles, or theidentification of the type of particle being analysed. In particular itis possible to assess more than one of these properties simultaneously.

At the same time, this invention allows these analysis to be carried outwith the use of considerably smaller amounts of chemicals than normallyare required to do these analysis. These chemicals are often consideredhazardous, either to humans and other living organism or to theenvironment. Furthermore, this invention presents a solution whichminimises the exposure of any hazardous sample or chemicals used for theanalysis by either allowing the analysis to be performed in a closedflow system or by the use of a sealed and disposable sample compartmentwhich contains all sample material and chemicals used for the assessmentand allows save transport of the sample and any chemicals.

The high cost as well as the mechanical complexity of the instrumentshitherto used for the routine assessment of the number of particles inliquid analyte material has made the instruments impractical to useroutinely under condition such as are normally present on dairy farms,on milk dairies, or in medical or veterinary clinics. Such analyses areof great interest, for instance, a dairy farmer can monitor the somaticcell count or bacterial count of an individual animal in order to followthe course of clinical or subclinical mastitis or infection, and tocontrol the cell count of the bulk milk delivered to the dairy, therebyminimising the use of antibiotics and preventing the economical penaltywhich is often a consequence when the cell count of bulk milk exceedspredefined limits.

Medical clinics are often in the need to know the count of one or moreparticles in blood, urine or other biological fluids such as somaticcells or bacteria, but since such analysis are usually carried out in acentral laboratory, this often delays the response of such analysis dueto transport of the sample.

It was found that this invention allows the analysis of various types ofbiological particles, such as DNA-containing particles, red blood cells,blood platelets, yeast cells, bacteria cells, lipid globules, proteinmicelles, dust particles, or polymer particles, these particles normallyfound in liquid biological analyte material such as milk, blood, urine,faces, salvia, inflammation, of either human or animal origin, orsamples originating from the petrochemical industry, the pharmaceuticalindustry, feed industry, food industry or the like. The method is alsowell suited for the detection of any other biological particle orfragments thereof, such particle being a part or a fraction of livingmatter and displaying properties which can be detected with thedetection of electromagnetic radiation.

This invention is particularly suited for the assessment of the numberof somatic cells in milk from human, cow, goat, sheep, buffalo or otheranimal. In particular, this invention is suited for the assessment ofthe number of somatic cells in milk during milking by integrating thesystem with the milking equipment, either in-line where the measurementis taken substantially from the milking system and analysed by aninstrument which is operated synchronised with the milking, or at-linewhere the sample is taken before, during or after milking and measuredon an instrument in manual operation, in particular it is well suited toobtain an estimate of the number of somatic cells when the purpose ofthe analysis is to control the number of somatic cells in the bulk ofmilk delivered to the dairy, for instance by directing any milk which isfound to have high cell count to a separate container or outlet.

Methods according to the invention are suited for the on-line or at-lineassessment of the number of somatic cells in milk when the purpose is toestablish information about the health status of animals, such as cows,goats, sheep or buffaloes, especially in connection with clinical orsub-clinical mastitis.

The method according to the invention is suited for the assessment ofthe number of somatic cells in milk when the objective of the analysisit to generate information used in a heard improvement scheme, or whenthe objective of the analysis is to obtain a quality parameter used in apayment scheme. These analyses are normally carried out on a centrallaboratory, by the use of complex instruments.

According to the invention, an array of detection elements can beutilised in combination with appropriate electronic components, toaccomplish the assessment of biological particles in a analyte materialby placing a portion of the analyte material in a sample compartment,the sample compartment in many embodiments of this invention being twowindows of glass, or other transparent material, separated by a spacerwith inlet and outlet which allows the sample to be replaced betweenmeasurements; in one embodiment, the sample compartment is a tube,substantially circular, or substantially elliptical in profile. Thepresence of a particle will normally cause the signal from a detectionelement to deviate from a normal level, e.g. a base-line level, eithertowards higher signal intensity or toward lower signal intensity, butfor the sake of clarity in the following it will be assumed that suchdeviation is toward higher signal intensity.

The present invention is based on the arrangement of the sample in sucha manner that it extends over a “window” of a substantial area anddetection of signals from the samples in the form of an “image” on anarray of detection elements, the array of detection elements comprisingindividual elements each of which is capable of sensing signals from apart of the sample window area, the array as a whole being capable ofsensing signals from substantially all of the sample window area, or atleast a well defined part of the sample window area.

As will appear from the following, the arrangement of the sample and thedetection elements in this way will allow the determination of thenumber of the particles per volume in a much more simple and economicmanner, while retaining a high accuracy of the determination. Also, aswill be explained in the following, the use of an array of detectionelements “observing” an exposed area of the sample makes it possible touse quite simple means for generating signals from the sample and quitesimple and sensitive detection means.

Thus, an aspect of the invention can be expressed as a method for theassessment of the number of particles in a volume of liquid samplematerial, the method comprising arranging a sample of the liquid samplematerial in a sample compartment having a wall defining an exposingarea, the wall allowing signals from the sample to pass through the walland to be exposed to the exterior, forming an image of signals from thesample in the sample compartment on an array of detection elements,processing the image on said array of detection elements in such amanner that signals from said particles are identified as distinct fromthe sample background, and, based on the signals from said particlesidentified assessing the number of particles in a volume of said liquidsample material.

Expressed in another and more general way, this aspect of the inventionrelates to a method for the assessment of at least one quantityparameter and/or at least one quality parameter of biological particlesin a liquid analyte material, comprising

applying a volume of a liquid sample representing the analyte material,or particles isolated from a volume of liquid sample representing theanalyte material, to an exposing domain from which exposing domainelectromagnetic signals from the sample in the domain can pass to theexterior,

exposing, onto an array of active detection elements, an at leastone-dimensional spatial representation of electromagnetic signals havingpassed from the domain, the representation being one which is detectableas an intensity by individual active detection elements, underconditions which will permit processing of the intensities detected bythe array of detection elements during the exposure in such a mannerthat representations of electromagnetic signals from the biologicalparticles are identified as distinct from representations ofelectromagnetic signals from background signals,

the size of the volume of the liquid sample being sufficiently large topermit the assessment of the at least one quantity parameter or the atleast one quality parameter to fulfil a predetermined requirement to thestatistical quality of the assessment based on substantially oneexposure,

processing the intensities detected by the detection elements in such amanner that signals from the biological particles are identified asdistinct from background signals,

and correlating the results of the processing to the at least onequantity parameter and/or the at least one quality parameter of theliquid analyte material.

The liquid sample representing the analyte material may be a liquidsample consisting of the liquid analyte material per se (optionally andoften preferably with added chemical substances facilitating theassessment, such as will be explained in the following), or it may be asample which has been derived from the liquid analyte material bydilution, concentration, extraction, or other modification. In thisconnection it is, of course, normally essential that there is anunambiguos correlation between the volume of the liquid samplerepresenting the liquid analyte material and the volume of the liquidanalyte material in question, so that the necessary correlation to aconcentration in the liquid analyte can be established. As mentionedabove, the liquid analyte material may in itself be a derivative ofanother material the properties of which are to be analysed using themethod of the invention; thus, e.g., the liquid analyte may be a liquidenrichment culture derived from a food product, e.g. poultry.

Alternatively, particles isolated from a volume of a liquid samplerepresenting the liquid analyte material may be the material from whichthe exposure onto the array of detection elements is made. This is thecase, e.g., when a liquid sample representing the liquid analytematerial has been filtered through a filter material, and the filtermaterial with the retained particles, often after addition of chemicalsfacilitating the assessment, cf. below, such chemicals having been addedbefore or normally after the filtration, is arranged in the domain fromwhich the exposure is made, normally a sample compartment suited forhousing the filter.

As mentioned above, the exposure of the electromagnetic signals havingpassed from the domain onto the array of detection elements willnormally correspond to forming an “image” of the domain (such as anexposing area of a wall part of a sample compartment) on atwo-dimensional array of detection elements, but it is also possible touse a one-dimensional spatial representation, obtained by suitableoptical means, in which case the array of detection elements need not bemore than one-dimensional, such as a linear array of detection elements.In special embodiments, a linear array of detection elements can also beused for receiving a two-dimensional image of electromagnetic radiation,provided the area of each element is sufficient to receive signals froma sufficient volume to allow the quality requirements to thedetermination.

The intensity detected by the array of detection elements may be acharge built up due to the electromagnetic radiation, or it may be,e.g., the intensity of a current passing through the individual elementas a result of the electromagnetic radiation.

The conditions of the exposure with respect to the various parametersinvolved, such as will be explained in greater detail below, are adaptedso that the intensities detected by the array of detection elements canbe processed, using suitable processing means, typically imageprocessing means and methods, in such a manner that the intensitieswhich have been detected as representations of electromagnetic signalsfrom the biological particles are identified as distinct fromrepresentations of background signals.

The size of the volume of the liquid sample on which measurement ismade, or from which the particles are isolated, should be sufficientlylarge to permit the assessment of the at least one quantity parameter orthe at least one quality parameter to fulfil a predetermined requirementto the statistical quality of the assessment based on substantially oneexposure. As will be explained in the following, it is a characteristicfeature of the present invention that it permits the gathering ofsufficient information in one exposure to allow a high statisticalquality in spite of the fact that the assessment can be performed in anextremely simple manner. One reason for this is that the method of theinvention is normally performed using much smaller enlargements of theimage projected onto the array of detection elements than has hithertobeen considered possible, and in some cases even reductions, in contrastto enlargements. For a number of applications, the degree of enlargementis just around 1:1, in contrast to most automated microscopy methodswhich use larger enlargements and several observations. In connectionwith the present invention, the term “substantially one exposure” is tobe understood as one exposure or in some cases just a few exposures suchas two, three or four exposures, but the by far preferred embodiment itso use just one exposure, such as is made possible by the invention. Theexposure may, under certain circumstances, be performed as a number ofsub-exposures before the intensity detected by the array elements isprocessed, but this is normally not necessary or preferred.

The formation of an image of the sample on the array of detectionelements may be performed by arranging the array of detection elementsin close contact or substantially in close contact with the exterior ofthe exposing wall of the sample compartment, or by using animage-forming means, such as a lens comprising one or several elements,arranged in the light path between the exposing wall of the samplecompartment and the array of detection elements.

The wall of the sample compartment defining an exposing area may be aflat or curved wall.

The sample in the sample compartment can be replaced by the means of aflow system, which is driven by a pump or a pressurised gas, preferablyair. In many embodiments of the present invention the flow in said flowsystem is controlled by one or more valves which can adjust the flowspeed of the sample.

In many preferred embodiments of the present invention the wall of thesample compartment is a plane wall, and the array of detection elementsis an array extending in a plane parallel to the plane of the wall.However, dependent on the manner in which the image of the sample isformed on the array of detection elements, the configuration of each ofthe exposing wall and the array may be designed in many different ways,such as where both the exposing wall and the array are configured assections of a circular cylinder, such as where the exposing wall isconvex and the array is concave with substantially the same radius,whereby they can easily be brought in contact or in substantial contactwith each other, or where both the exposing wall and the detection arrayare concave, and a lens is used for formation of the image of the sampleon the array. Many other configurations are, of course possible, such aswhere both the exposing wall and the array are sections of spheres, etc.

The sample compartment may be a chamber which can easily be removed fromthe instrument when a new sample or sample material is to be measured.Such removable sample compartment is preferably used for a limitednumber of measurements and preferably only one. Apart from allowing amore simple mechanical construction of an instrument with the absence ofany flow system, one advantage of such removable sample compartment isthat it can contain the sample in a closed container before, during andafter analysis, thus allowing more safe handling of hazardous material.In many embodiments of the present invention such removable samplecompartment can, prior to the introduction of any sample material,contain one or more component or device used for chemical or physicalmodification of the sample prior to analysis.

Electronical devices or a computer equipped with suited software can beused to condition a signal which originates from any detection elementused, preferably in such a way as to make the quantification of thesignal from any detection element more reliable or less time consuming,for instance by converting one type of signal to another signal suitedfor processing, and/or by providing means for the amplification of thesignal. Often it is preferred that the signal from any detection elementis adjusted for any bias, and/or for any variation in sensitivity whichmight be present in the signals, this adjustment preferably beingperformed by taking into account information from neighbouring detectionelements, or by using similar information from a previous measurement.Another useful property of such signal conditioning is the conversion ofa substantially analogue signal to a digitised value which is bettersuited for further processing using a digital data processing system;such digitalisation could be a threshold-like activation of two or moreoutput lines in such a way that the input level of any signal wouldcause a change the status of these output lines, preferably in such away that the level of the input signal could be estimated. A preferredmethod of digitalisation is one which allows the level of the inputsignal to be converted to a number according to the binary numbersystem.

It is often preferred that the digital representation of the level ofany input signal produces a substantially linear function, and in manypreferred embodiments of this invention it is preferred that the digitalrepresentation produces a substantially non-linear function, forinstance a logarithmic function, such non-linear function beingpreferred when the dynamic range of the input level is high.

In some implementations of this invention, it is preferred to use aone-dimensional array of detection elements, preferably included in onechip, the identification of a particle present in the sample which ismeasured being done by comparing the level of signal from each detectionelement with a predefined level, or preferably to a level which isestimate on the basis of the signals from neighbouring detectionelements, preferably on the basis of the signals from previousmeasurements, and if a signal is found to be above this discriminatinglevel it is assumed that a particle was present, and a counter isincremented accordingly. Furthermore, it is possible to detect thepresence of two particles measured at once for instance by comparing theintensity of a signal to a known or determined limits in such a way thatsignals above such limit indicate the presence of two particles. Morethan one such limit can be used to identify any situation where three,four or more particles are present, or an empirical or theoreticalrelationship can be constructed between the total number of particlespresent, the possibilities of signals from two or more particles beingdetected simultaneously by a detection element.

As mentioned above, it is often preferred that an optical system is usedto focus any signal from the sample onto the detection elements, and insome cases, it is preferred that such focusing produces an image of aparticle with an average size which is of about the same size as thedetection elements used, and in certain cases preferably smaller, suchthat the image of the entire particle is substantially within theboundaries of the detection element.

In other embodiments of this invention, similar to the one describedabove using a one dimensional array of detection elements or atwo-dimensional array of detection elements, it is preferred that anoptical system is used to focus any signal from the sample onto thedetection elements in such a way as to produce an image which is of thesame size as the detection elements used, or in some cases preferablygreater, the method being used to identify the presence of a particletaking into account also the extension of the particle in the dimensionalong the row of detection elements as well as the height of themeasured signal from each detection element. Such embodiment of thisinvention allows the estimation of some morphological properties of theparticles which are measured, such as the size. Also under thoseconditions it is possible to detect the presence of two ore moreparticles which are focused on substantially the same detectionelements, for instance by classifying the signal intensity.

It was surprisingly found that a one dimensional array of detectionelements, where the width of the array of detection elements wasconsiderably greater than the height of each detection element, onecommercially available from Hamamatzu (S3902-128Q), could be excellentlyused for the assessment of the number of particles and thus enabling thedetection of signals from a greater volume of the sample in eachscanning of the detection elements. Furthermore, it was discovered thatthe use of even a focusing device which distorts the dimensions of theimage, relative to the original, in such a way that for instance theimage of a circle has a shape which is similar to an ellipse, also gavesimilar advantage as the use of detection elements with great height,and further it was found that with a combination of the above-mentioneddetection elements and a distorting focusing device it was possible toobtain a useful assessment on a large detection volume.

The use of a series of one dimensional arrays of detection elements,preferably incorporated in a single chip, is often found to be useful inthe assessment of biological particles present in a sample, onecommercially available charge coupled device (CCD) is available fromSony (ICX 045 BL). Another array of detection elements suited for manyembodiments of this invention is an image sensor based on CMOStechnology which makes detection possible with the use of limitedelectrical effect, as well as offering on-chip integration with otherCMOS based technologies such as signal condition and signal processing,one such has been demonstrated by Toshiba comprising 1318×1030 elementseach about 5.61 μm×5.6 μm in size using only 30 mW effect in use.

The assessment of biological particles in a sample can be performed bytreating each line of such two dimensional array of detection elementsin substantially the same manner as an array of one dimensionaldetection elements.

Some embodiments of this invention allow the simulation of highdetection elements by the electronical or computational addition ofinformation from two or more lines of detection elements into one arrayof information which is thereafter treated in the substantially the samemanner as a single one dimensional array of detection elements, thusallowing substantially simpler and less time consuming interpretation ofthe measured information.

In some embodiments of this invention the assessment of the number ofparticles in a first line of detection elements is based on any results,such as position and/or intensities observed in a second line ofdetection elements already being processed, thus allowing the correctionof signals which extend across two or more lines of detection elements.

The inclusion of a focusing device for the focusing of a signal from thesample onto the detection elements in such a manner as to maximise thecollection angle, the collection angle being defined as the full planeangle within which a signal is detected, has in many situations beenfound to give improved conditions for an assessment. Surprisingly it wasfound that such a wide collection angle, even to the extent that theobjective used in the focusing distorted the aspect ratio of the imageof any particle differently across the plane in which the detectionelements were placed, or produced variation in the focusing across thesample being analysed, or reduction of the focusing quality, could beused in the assessment of the number of particles.

It is possible to make the assessment of biological particles in asample by using a calculation mean, preferably a digital computer, onecommercially available from Analogue Devices (ADSP 2101), equipped withstorage capacity which can onbly store information in amountsubstantially equivalent to a small fraction of the total number ofdetection elements, the assessment of the number of objects then beingbased on substantially real time processing of data, preferably in sucha way that the measured information from each detection element, or aline of detection elements, or two or more lines of detection elements,is used for the assessment, substantially without any delay, such as adelay which would otherwise be caused by storing the measuredinformation.

However, it is often preferred to store substantially all measuredinformation by the use of a first calculation mean, preferably a digitalcomputer, before the processing of the information by a secondcalculation mean, preferably a digital computer, and thus allowing themeasured information to be processed at substantially the same rate itis obtained, but with a substantial time delay between the measurementof any information and the processing of the same information;preferably, this is accomplished by using only one calculating mean,preferably a digital computer, equipped with enough resources toaccomplish the task.

When using a sample compartment used for the analysis of more than onesample material, for instance when the sample is introduced by means ofa flow system, it is often found that one or more of the particles ofinterest, or fractions of particles, adhere to the sample compartment insuch a way that the flow used to replace the sample material is notcapable of removing said adhering particles. Thus if such adheringparticles are situated in a place which is exposed to the sensingdevice, it will be included in two or more observation although thesample has been substantially replaced between observations. In manyembodiments of the present invention the influence of said adheringparticles on the observation can be substantially eliminated bycombining two observations in such a way that the result from a firstobservation is adjusted by the result from a second observation, saidsecond observation being one of many observations taken prior to saidfirst observation or a combination of more than one of many observationstaken prior to said first observation, preferably an observation takensubstantially immediately prior to said first observation, saidadjustment being a simple subtraction of said second observation fromsaid first observation. The result of said adjustment then containsinformation where any objects present in said first observation havepositive intensity, and any object present in said second observationhas negative intensity and any object present in both first and secondobservation have substantially zero intensity. The task of any methodused for the assessment of the number of objects or the determination ofany morphological properties of an object is then to only treat thoseintensities which have substantially positive values. In a similar wayit is possible to analyse the results of two or more observation takenfrom different samples from the same sample material by combining thoseobservations as described above and subsequently to analyse both thepositive and negative signals, for instance by treating all signals asbeing positive. In this way it possible to analyse 2, 4, 6, 8 or moreobservations simultaneously for instance in situations where the effortof analysing an observation is greater than the effort of making anobservation.

This invention allows the sample material to be a substantially aqueoussolution, or substantially organic solution, or a mixture of two or moreimmiscible phases, some of which can be liquid, some of which can besolid and some of which can be a suspension, into which the particles ofinterest are suspended. In many preferred embodiments of this inventionthe sample material to by analysed has been modified or its chemical orphysical properties substantially changed compared to the analytematerial by either the addition of, or the removal of one or morecomponents, or by introducing the sample to one or more chemical,mechanical or physical treatments prior to analysis. Preferably theeffect of any such alteration or modification is the enhancement of anymeasurable signal used for the analysis, or a suppression of anyinterfering phenomenon, or it has the effect of prolonging the workinglife of the sample.

It is often preferred that the signal which is detected is aphotoluminescence signal, originating from a molecule, or a fraction ofa molecule having fluorophore properties, naturally contained within oron the particle which is measured.

The particles which are to be detected are often “coloured” with one orseveral molecules which bind to the particle, are retained within theparticle, or otherwise interact with the particle, the effect of this“colouring” being the enhancement of any signal for the particle, orbeing the direct source of a signal which thereby can be used to detectthe particle.

In many aspects of the invention the effect of the “colouring” is tocause, or enhance, the attenuation of electromagnetic radiation such asvisible light, or preferably to cause, or enhance, the emission ofelectromagnetic radiation such as chemiluminescence, orphotoluminescence, e.g. fluorescence or phosphorescence, when exitedwith radiation which is substantially higher in energy that the emittedphotoluminescence. One such “colouring” is the addition of EthidiumBromide (EtBr) to the sample, where EtBr interacts with DNA materialpresent in the sample, giving rise to fluorescence at approximately 605nm when exited with light at approximately 518 nm (Handbook ofFluorescent Probes and Research Chemicals, page 145). This makes itpossible, in the combination with the appropriate set of opticalfilters, to count a DNA-containing particle where EtBr can interact withthe DNA, such particles are for instance cells containing DNA, inparticular DNA containing somatic cells or bacteria, such as thosepresent in milk, blood or urine.

It was surprisingly found that it was possible to use concentrations offluorophore which were substantially lower than those normally used insystem, often less than 1/10th or 1/100 or even less than 1/1000; inparticular this is advantageous where added fluorophore exhibitsrelatively similar properties in free form as in bound form, with regardto intensity and wavelength characteristics. As expected, such conditioninherently reduce any signal emitted from a coloured particle, butsurprisingly it was found that the ratio of the signal intensity inbound form to free form shifted in favour of bound signals. Inparticular it was found that a level of signal from fluorophore in freeform in the sample which was comparable, and preferably less, inintensity to any random electronical signal (noise) and/or comparable inintensity to, and preferably less than, any other interfering signal wasto be preferred.

It is often preferred that the liquid in which particles which are to bemeasured are suspended, is substantially at stand-still, wherestand-still is defined as the situation where at least a part of theimage of a particle does not move any more than it is containedsubstantially within the boundary of the same detection elements duringone measurement period. The stand-still situation is preferably suchthat at least a part of the image of a particle does not move any morethan it is contained substantially within the boundary of the samedetection element during at least two measurement periods, thus allowingthe detection of any weak signals which might indicate the presence of aparticle.

In other embodiments of this invention, normally less preferred, theliquid in which particles which are to be measured are suspended, issubstantially moving during measurement, in such a way that at least apart of the image of a particle gives rise to signal in two or moreadjacent detection elements during one measurement period, or in such away that at least a part of the image of a particle gives rise to signalin two or more adjacent detection elements during at least twomeasurement periods.

The liquid in which particles which are to be measured are suspended canbe moving in more than one direction during measurement, for instance bycontrolling two sources of force, preferably which can be appliedperpendicular to each other, thus giving the opportunity to move thesample in a predefined pattern, which can be used to improve theperformance of any image processing device used to analyse the measuredsignal.

It is possible to perform more than one measurement and thus allowing amore accurate and/or sensitive assessment of the number of particles,for instance by measuring the same portion of the sample more than onceand combining the results in order to improve the signal to noise ratio,and/or to measure more than one portion of the sample in order toincrease the total number of particles which are counted to reduce theerror in the assessment since the error in the particle count willnormally follow count statistics where the relative error is expected tobehave similar to one over the square root of number of counts. However,it is a characteristic feature of the present invention that its generalcharacter of detection based on a relatively large sample volume givinga large amount of information makes it possible to meet a predeterminedstatistical standard based on substantially one exposure.

In some embodiments of this invention the number of measurements whichare taken is defined by a real time estimate of the number of particlesalready counted thus performing relatively fewer measurements when thesample contains a high number of particles and relatively moremeasurements when the sample contains a low number of particles,preferably by defining an approximate lower limit for the total numberof counted particles in such a way as an appropriate accuracy in themeasurement is obtained.

It is possible to assess the biological particles in a relatively shorttime thus allowing a high number of samples to be analysed per hour,often more than 400, and even as many as 1000 or more analysis per hour.In many preferred embodiments of this invention an even higher numberanalysis per hour is achieved by including more than one measurementunit, the measurement units working in parallel in a single instrument.

In many embodiments of this invention the signals which are detected areattenuation of electromagnetic radiation, for instance caused byabsorption or scattering, and in many preferred embodiments of thisinvention the signals which are detected are emitted from the particlesor the samples, for instance emission of photoluminescence (e.g.fluorescence and/or phosphorescence) or raman scatter, and in otherembodiments of this invention the signals which are detected are causedby scatter.

Often more than one of the previously mentioned signals are detectedsimultaneously thus allowing more accurate or sensitive assessment ofthe number of particles or the assessment of any morphological propertyor to allow classification of a particle present in the sample,preferably by the use of more than one set of detection elements.

A monochromatic device can be used to separate electromagnetic radiationinto one or more wavelength components before one or several of thesewavelength components are transmitted onto the sample either one at atime or more than one at a time, preferably when more than onewavelength component is transmitted onto the sample simultaneously thewavelength components are transmitted onto different portions of thesample thus giving an opportunity to obtain qualitative as well asquantitative information about particles in the sample. This is inparticular of interest when the sample contains particles which responddifferently to different wavelength components.

Light which can be transmitted onto the sample can be focused by afocusing system, comprising one or more lenses. The effect of such afocusing system is often to increase the effective efficiency of thelight source. As light source it is possible to use a thermal lightsource, such as a halogen lamp, or a gas lamp such as a xenon lamp, alight emitted diode, a laser or a laser diode. It is often preferred touse more than one light source for the purpose of increasing the flux oflight onto the sample, for instance by using two or more light emittingdiodes. It is also possible to use more than one light source where someof the light sources have different electromagnetic properties.

A monochromatic device can be used to separate electromagnetic radiationemitted from, or transmitted through the sample into one or morewavelength components before such electromagnetic radiation is detectedby a detection element, either in such a way that one wavelength ismeasured at a time or in such a way that more than one wavelengthcomponents are measured at a time. This is in particular of interestwhen the sample contains particles which respond differently todifferent wavelength components for instance when a particle is capableof emitting photoluminescence with different properties dependent on thenature of the particle. This effect can also be produced by the use ofmore than one type of light source which have different wavelengthcharacteristics, preferably in combination with a monochromatic device.

In many preferred embodiments of this invention electromagneticradiation, such as UV or visible light is transmitted onto the sample,in order to give rise to photoluminescence, in a set-up where the lightsource, the sample compartment and the detection elements all aresituated approximately on the same axes, preferably where the samplecompartment is situated between the light source and the detectorelements. Surprisingly it was found that under these conditions it waspossible to remove substantially all the excitation light which wastransmitted through the sample by means of filters, even in situationwhere high amounts of energy were used for the excitation. Further inmany preferred embodiments of this invention it was found that it waspossible to increase the efficiency of the electromagnetic radiationused for excitation by placing a reflecting device between the samplecompartment and the detector which could reflect at least a portion ofthe energy transmitted through the sample compartment back towards thesample compartment, preferably where at least one of the surfaces whichdefine the sample compartment was reflecting, preferably this reflectingdevice is one which has different reflectance properties at differentwavelength, preferably in such a way that it is substantiallytransparent to the photoluminescence signal. One such reflecting deviceis a dichroic mirror.

It is often preferable to use one or several state of the art imageprocessing techniques, such as 2 dimensional filtering or imageidentification, to assess the number of particles, or any morphologicalproperty of a particle.

As mentioned above, it is a particular feature of the invention thatcompared to traditional microscopy methods, the enlargement is fromrelatively small to very small. Thus, it is often preferred that thespatial representation exposed onto the array of detection elements issubject to such a linear enlargement that the ratio of the image of alinear dimension on the array of detection elements to the originallinear dimension in the exposing domain is smaller than 40:1, normallyat the most 20:1, preferably smaller than 10:1 and in many cases even atthe most 6:1 or even smaller than 4:1.

The enlargement is suitably adapted to size of the particles to bedetermined. Thus, for example, when the particles the parameter orparameters of which is/are to be assessed are of a size of between ⅓ μmto 3 μm, the above-mentioned ratio is preferably in the range between40:1 and 1:10, more preferably in the range between 20:1 and 1:10, suchas in the range between 10:1 and 1:10. Inmost embodiments which haveproved to give excellent results in practice, the ratio is in the rangebetween 6:1 and 2:1.

When the particles the parameter or parameters of which is/are to beassessed are of a size between 3 μm and 100 μm, the above-mentionedratio is normally in the range between 3:1 and 1:100, preferably in therange between 2:1 and 1:100. In many practical embodiments, the ratiowill be in the range between 2:1 and 1:2. It can be interesting, inparticular with small high precision detection elements, to work withvery small rations, such as in the range between 1.4:1 and 1:100, e.g.,in the range between 1:1 and 1:100.

Another way of expressing the ratio at which the image should preferablybe formed on the array is to consider the imaging of the individualparticle on the detection elements. It is often preferred that theindividual particles the parameter or parameters of which is/are to beassessed are imaged on at the most 25 detection elements, in particularon at the most 16 detection elements and more preferred at the most 9detection elements. It is even more preferred that the individualparticles the parameter or parameters of which is/are to be assessed areimaged on at the most 5 detection elements, or even on at the most 1detection element. The larger number of elements per particle willprovide more information on the individual particles, while the smallernumber of elements per particle will increase the total count that canbe made in an exposure.

As mentioned above, it is one of the characterising features of thepresent invention that a relatively large volume of sample can beexposed to the detection array. The sample is contained in the interiorof the domain or sample compartment, which normally has an averagethickness of between 20 μm and 2000 μm, usually between 20 μm and 1000μm and in many practical embodiments between 20 μm and 200 μm. Normally,the domain or sample compartment has dimensions, in a directionsubstantially parallel to the array of detection elements, in the rangebetween 1 mm by 1 mm and 10 mm by 10 mm, but is will be understood thatdepending on the design, it may also be larger and, in some cases,smaller.

The volume of the liquid sample from which electromagnetic radiation isexposed onto the array is normally in the range between 0.01 μl and 20μl. When the particles the parameter or parameters of which is/are to beassessed are of a size of between ⅓ μm to 3 μm, the volume of the liquidsample from which electromagnetic radiation is exposed onto the array isnormally in the range between 0.01 μl and 1 μl. When the particles theparameter or parameters of which is/are to be assessed are of a size ofbetween 3 μm to 100 μm, the volume of the liquid sample from whichelectromagnetic radiation is exposed onto the array is normally in therange between 0.04 μl and 4 μl.

As mentioned above, the sample is preferably at stand still during theexposure.

However, in another embodiment, the sample in the domain or samplecompartment is moved through the domain or sample compartment during theexposure, and the exposure is performed over a sufficiently short periodof time so substantially obtain stand still condition during theexposure. In either case, there is a close control of the volume of thesample from which the exposure is made, which is one very preferredfeature of the present invention.

When at least a major part of the electromagnetic radiation emitted fromthe sample during exposure originates from or is caused byelectromagnetic radiation supplied to the sample from a light source, itis highly preferred at least a major part of the radiation from thelight source having a direction which is transverse to the wall of thesample compartment or a plane defined by the domain, such assubstantially perpendicular to the plane defined by the domain (or anincrement plane if the compartment wall is curved), or betweenperpendicular and 10 degrees, preferably between perpendicular and 20degrees, more preferably between perpendicular and 30 degrees and stillmore preferably between perpendicular and 45 degrees. This is incontrast to the case where the radiation enters from an edge, parallelto the plane of the sample compartment, which is considered highlydisadvantageous as it will, for many sample types, give rise tosufficient illumination of only a small rim part of the sample.

As mentioned above, the size of the volume is suitably adapted to thedesired statistical quality of the determination. Thus, where thedetermination is the determination of the number of particles in avolume, or the determination of the size and/or shape of particles, thesize of the volume of the liquid sample is preferably sufficiently largeto allow identification therein of at least two of the biologicalparticles. More preferably, the size of the volume of the liquid sampleis sufficiently large to allow identification therein of at least fourof the biological particles. This will correspond to a repeatabilityerror of approximately 50%. Still more preferably, the size of thevolume of the liquid sample is sufficiently large to allowidentification therein of at least 10 of the biological particles. Thiswill correspond to a repeatability error of approximately 33%. Even morepreferably, the size of the volume of the liquid sample is sufficientlylarge to allow identification therein of at least 50 of the biologicalparticles. This will correspond to a repeatability error ofapproximately 14%. Evidently, where possible, it is preferred to aim atconditions where the size of the volume allows identification of evenhigher numbers. Thus, when the size of the volume of the liquid sampleis sufficiently large to allow identification therein of at least 100 ofthe biological particles, it will correspond to a repeatability error ofapproximately 10%, and when the size of the volume of the liquid sampleis sufficiently large to allow identification therein of at least 1000of the biological particles, it will correspond to a repeatability errorof as low as approximately 3%.

Expressed in another, more specific manner, one main aspect of thepresent invention is defined as a method for the assessment of at leastone quantity parameter and/or at least one quality parameter ofbiological particles in a liquid analyte material, comprising

applying a volume of between 0.01 μl and 20 μl of a liquid samplerepresenting the liquid analyte material, or particles isolated from avolume of a liquid sample representing the liquid analyte material, toan exposing domain from which exposing domain electromagnetic signalsfrom the sample in the domain can pass to the exterior,

exposing, onto an array of active detection elements, an at leastone-dimensional spatial representation of electromagnetic signals havingpassed from the domain, the representation being one which is detectableas an intensity by individual active detection elements, underconditions which will permit processing of the intensities detected bythe array of detection elements during the exposure in such a mannerthat representations of electromagnetic signals from the biologicalparticles are identified as distinct from representations ofelectromagnetic signals from background signals, the conditionsinvolving such a linear enlargement that the ratio of the image of alinear dimension on the array of detection elements to the originallinear dimension in the exposing domain is smaller than 10:1, and suchthat the individual particles the parameter or parameters of whichis/are to be assessed are imaged on at the most 25 detection elements ofthe array of detection elements,

the sample in the domain or sample compartment being at stand stillduring the exposure, and in the case where at least a major part of theelectromagnetic radiation emitted from the sample during exposureoriginates from or is caused by electromagnetic radiation supplied tothe sample from a light source, then at least a major part of theradiation from the light source having a direction which is transverseto the wall of the sample compartment or a plane defined by the domain,

processing the intensities detected by the detection elements in such amanner that signals from the biological particles are identified asdistinct from background signals,

and correlating the results of the processing to the at least onequantity parameter and/or the at least one quality parameter of theliquid analyte material.

As mentioned above, the signal which is detected by the detectingelements originates from one or several types of molecules of typeswhich bind to, are retained within, or interact with, the biologicalparticles, such molecules being added to the sample or the isolatedparticles before or during exposure, the molecules being moleculesgiving rise to one or several of the following phenomena: attenuation ofelectromagnetic radiation, photoluminiscence when illuminated withelectromagnetic radiation, scatter of electromagnetic radiation, ramanscatter. In the presently most preferred embodiments an effective amountof one or more nucleic acid dyes and/or one or more potentiometricmembrane dyes is added.

The duration of the exposure is in normally the range from 100milliseconds to 5 seconds, in particular in the range of 0.5 to 3seconds. The exposure may be performed as multiple exposures before theintensities detected by the detection elements are processed, but it isnormally preferred that the exposure is performed as a single exposure.

A number of embodiment and variants of the invention appear from thefigures and examples which follow, as well as of from the followingdetailed description of embodiments.

With these and other objects in view, which will become apparent to oneskilled in the art as the description proceeds, this invention residesin the novel construction, combination, arrangement of parts and methodsubstantially as hereinafter described, and more particularly defined bythe appended claims, it being understood that changes in the preciseembodiments of the herein disclosed invention are meant to be includedas come within the scope of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one embodiment of this invention, particularly suitedfor the assessment of particles by the use of fluorescence.

FIG. 2 illustrates the effect of varying the initial concentration offluorescent labelling dye.

FIG. 3 illustrates the possible removal of systematic bias by thesubtraction of measured signals.

FIG. 4 illustrates an optical arrangement allowing collection of signalswith a collection angel of approximately 40 degrees.

FIG. 5 illustrates an optical arrangement allowing collection of signalswith a collection angle of approximately 70 degrees.

FIG. 6 illustrates components used for a flow system.

FIG. 7 illustrates a disposable measurement and sampling cell for theassessment of the number of somatic cells in a volume of milk.

FIG. 8 illustrates an instrument for the assessment of the number ofsomatic cells in a volume of milk.

FIG. 9 is a graph of the assessment of the number of somatic cells in111 of milk plotted against results obtained by a FossoMatic routineinstrument.

FIG. 10 is a graph of the number of counted objects in a milk sample vs.the concentration of the fluorochrom.

FIG. 11 illustrates the effect of processing two dimensional image.

FIG. 12 illustrates the representations of intensities in themeasurement of bacteria in an aqueous sample.

One aspect of the invention consems a method for compression ofintensity information representing distinct objects scattered over anarea, an object being represented by a variation in the intensityinformation.

The information existing in the form of varying degrees of measurableintensity of a physical property distributed over a confined areadivided into sub-areas, each of which sub-areas having assigned theretoan index uniquely identifying the sub-area. According to the inventionthe method comprising determination of the intensity of the physicalproperty.

One such method of determining the intensity of the physical propertycould be a read-out of the electrical signal from an array of detectionelement such as CCD-elements exposed to electromagnetic radiationradiated from the objects scattered oven an area. Another such methodcould be by exposing a photografich film to electromagnetic radiationradiated from the objects scattered over an area, thereby generating a“picture” of the intensity of the physical property which then could bedigitized in such a manner that for instance a number is assigned to adistinct interval of intensities represented by for instance gray-scalein the “picture”

When the determination of the intensity has been done the compressioncould be performed by the following:

-   -   a) defining a sub-area of interest situated in a group of        sub-areas comprising of at least 2×2 sub-areas situated adjacent        to each other,    -   b) evaluating in said sub-area of interest at least one        directional derivative(s) of the measurable intensity in the        sub-area of interest with respect to predetermined geometrical        direction(s) in the plane of the confined area, the directional        derivative(s) is (are) based on measurable intensities in        sub-areas situated adjacent to or in proximity of the group of        sub-area,    -   c) based on the evaluation of the at least one directional        derivative an attribute is assigned to the value assigned to        said sub-area of interest; the attribute represent an adjusted        measurable intensity and/or information(s) related to a        predetermined strategy for adjustment of the measurable        intensity in the sub-area of interest or sub-areas situated        adjacent to or in proximity to the sub-area of interest.        The steps a)-c) can be performed for substantially all sub-areas        of the confined area.        The evaluation of the directional derivatives is used to give an        indication of where the center of an object is situated relative        to the confined area and to give a measure of which of the        sub-area in the confined area which is subject to be a sub-area        in which information from neibouring sub-areas should be        assigned to. Such information could be the intensity        information.        The steps a)-c) can be repeated succesively for some of the        sub-areas or for all of the sub-areas.        The use of the attribute enables the possibility of storing        information of the about the sub-area of interest such that an        eliptical, hyperbolical or and parabolic method (in a        mathematical understanding) can be used as a strategy for        distribution of intensity information from some of sub-areas to        selected sub-areas in the confined area, the number of selected        sub-areas are lower in number than the sub-areas contained in        the confined area.

EXAMPLE 1 Detection of Fluorescence Signals from Ethidium Bromide (EtBr)Bound to DNA in Somatic Cells in Milk at Different Initial ConcentrationLevels of Ethidium Bromide

The sample material was cow bulk milk. To each of three portions of thesame sample material, used for the below Experiments A, B and C, wasadded a buffer in the ratio of two parts by volume of buffer solution toone part by volume of milk. The buffer solutions were identical exceptthat they contained different amounts of EtBr. The buffer solutions wereprepared according to the guidelines of International IDF standard 148A:1995—“Method C, concerning Flouro-Opto-Electronic Method” (Experiment A,EtBr concentration 33 μg/ml); for Experiment B, the concentration ofEtBr was 10% of the prescribed amount, and for Experiment C, it was 1%of the prescribed amount.

The resulting sample materials were measured in a set-up as follows (cf.FIG. 1): A halogen lamp 101 of type OSRAM (41890 SP 12V, 20 W, 10 degreereflector) was used as a light source emitting electromagnetic radiationonto the sample contained in a sample compartment 104 through acollecting lens 102 and through an optical filter 103 selectivelytransmitting light in the waveband between 400 and 550 nm (Ferroperm SWP550). Any fluorescence signal originating from the sample was focusedusing a lens 105 with a collection angle of approximately 10 degrees andproducing an image which was approximately 4 times larger than thesource on a two-dimensional array of detection elements 107, constitutedby a CCD of the type Loral Fairchild (CCD 222). An optical filter 106selectively transmitting light in a waveband between 600 and 700 nm(Schott OG590 and KG5, thickness 3 mm) was inserted between the samplecompartment and the array.

The final concentration of EtBr in each experiment and the operation ofthe light source and the detector elements was as follows: ExperimentEtBr (μg/ml) Lamp (Volt) CCD Integration time (ms) A 33 12 800 B 3.3 12800 C 0.33 13 1600

The data from the two dimensional array of detection elements wasdigitised and collected on a computer (not shown) for later analysis.

Results

Data from the two dimensional array of detection elements was used toproduce images in which the intensity detected by each element isillustrated as height over a graphic representation of the array. Anillustration of this type of typical signals from each experiment isshown in FIG. 2, where FIG. 2A is a representation of intensities asobserved in experiment A, FIG. 2B is a representation of intensities asobserved in experiment B, and FIG. 2C is a representation of intensitiesas observed in experiment C. Peak-like structures in the figures,distinct from representations of electromagnetic signals from the samplebackground, are representations of EtBr bound to DNA in somatic cellscontained in the milk samples.

In all cases the figures are the numerically positive result of thesubtraction of one measurement from the sample, from another measurementof a different portion of the sample, by using the formula:Signal_((ij))=ABS(meas1_((ij))−meas2_((ij))), where i and j refer to therow and column of the CCD, thus suppressing any systematic bias of themeasurement system.

In experiment A, illustrated in FIG. 2A, the signal intensity was suchthat a majority of the cells showed signals which caused charge overflowon the CCD, resulting firstly in the cut-off of the signal due to thefact that the signal was outside the range of the detector elements, andsecondly in broadening of the signal top due to charge transfer fromoverloaded detection elements to neighbouring detection elements. Inaddition it is obvious that the variation in the signals of thebackground is high, presumably due to interaction between free EtBr andthe sample matrix (for instance fat globules and protein micelles).

FIG. 2B illustrates typical signals as observed in Experiment B.Experiments A and B were identical apart from the concentration of EtBrused, and the beneficial effect of the lowering of the EtBrconcentration on the signal intensity and signal broadening is evident.In addition, the random variation in the background is about ½ of thevariation observed in experiment A.

FIG. 2C illustrates typical results from Experiment C. In thisexperiment, the intensity of excitation light as well as the integrationtime of the detection elements were increased. The result fromexperiment C is that the signals are considerably weaker than inexperiment B, with a background signal of similar magnitude.

Conclusion

The above results illustrate that it is possible to detect signals fromsomatic cells using concentrations of EtBr which are considerably lowerthan concentrations normally used for the fluorescence detection ofDNA-containing particles.

One preferred embodiment of this invention is based on an optical systemwhich has a collection angle of between 40 and 70 degrees, as comparedto the 10 degrees used in the present example; and this will result inthe collection of approximately 10 to 300 times as much energy, makingit possible to reduce the concentration of the reagent even further.

EXAMPLE 2 Removal of Signal Bias by Combination of Measurements from aLinear Array of Detection Elements

Removal of systematic signal bias can be of interest in the processingof measured signals. In the present example a linear array of detectionelements of the type Hamamatsu (S3902-128Q) was used in an arrangementsimilar to the one illustrated in FIG. 1. Under the conditions used, thearray of detection elements gave a readout which had a systematic biasbetween detection elements with even index and detection elements withodd index. A series of 2 measurements was carried out using water assample material.

Results

FIG. 3 shows the results of the measurements of water. FIG. 3A shows theresult from the first measurement after the measurement had beenadjusted for the mean bias. From FIG. 3A it is apparent that there is aclear difference in the signal intensity of odd and even detectionelements, in that elements with an odd index have generally lowersignal. FIG. 3B shows the result of scan 1 after the results from scan 2have been subtracted. What is apparent is that the systematic effect ofodd and even detection elements has been substantially removed,resulting in a signal with a baseline which can be expected to havevariations of more random character; the amplitude of this noise can beexpected to have an amplitude of approximately 1.41 the amplitude of anyrandom noise present in one measurement.

Conclusion

The conclusion from the above result is that it is possible to remove asystematic bias by subtracting one measurement from another. In additionto variations in the detecting system, systematic bias can be caused bymany other factors, such as particles adhering to the wall in a flowsystem, variations in the intensity of excitation light from a lightsource consisting of a plurality of elements such as light-emittingdiodes, etc. Compensation for systematic bias, performed, e.g., asillustrated in the present example and in Example 1, will enhance thedistinction between representations of electromagnetic signals from thebiological particles and representations of electromagnetic signals fromthe sample background. However, for many applications, the inherentdistinction obtained using the method of the present invention will beadequate or more than adequate even without a compensation forsystematic bias. The use of disposable sample compartments used onlyonce will rule out any problems ascribable to adhering particles in aflow system.

EXAMPLE 3 Optical Configuration for Wide Angle Collection of Signal froma Sample

It can be demonstrated that the intensity of any signal collected from asample is dependent on the square of the collection angle. Inconventional automated microscopy, the collection angle is at the most20 degrees and normally considerably lower, such as 1-5 degrees. Becauseof the low magnification (or no magnification) which can be usedaccording to the present invention, and the robust processing madepossible thereby, a much larger collecting angle can be used. In thepresent example two different optical arrangements are used to obtain acollection angle of approximately 40 and approximately 70 degrees,respectively.

FIG. 4 illustrates an optical arrangement which produces a collectionangle of approximately 40 degrees when collecting a signal from a samplecompartment 401 and projecting it onto detection elements 404, by usingtwo achromatic lenses, one 402 of the type Melles Griot 01 (LAO 014:F=21 mm, D=14 mm) and another one 403 of the type Melles Griot 01 (LAO111: F=80 mm, D=18 mm).

FIG. 5 illustrates an optical arrangement which produces a collectionangle of approximately 70 degrees when collecting a signal from a samplecompartment 501 and projecting it onto detection elements 506, by usingone immersion lens 502 with radius of approximately 5 mm and width ofapproximately 8.3 mm, and one aplanatic meniscus lens 503 with oneradius of approximately 12.5 mm and one radius of approximately 10.5 mm,and two identical achromatic lenses 504 and 505 of the type Melles Griot01 (LAO 028: F=31 mm, D=17.5 mm).

EXAMPLE 4 Components of a Disposable Measurement and Sampling Unit

The components of a flow system which can be used for the assessment ofbiological particles according to principles of the present inventionare given in FIG. 6. The components in FIG. 6 are as follows: An inlet601 where the sample is introduced to the flow system, a pump 602situated upstream from the sample compartment, a valve 603 controllingthe inlet flow of the sample, means 604 allowing introduction of one orseveral intentionally added chemical components, means 605 which allowthe mixing of the sample and one or several chemical components and/orany other mechanical or physical operation such as retaining particles,a sample compartment 606, a valve 607 controlling the flow from thesample compartment, a pump 608 situated downstream of the samplecompartment, an outlet 609 from the flow system and a unit 610 housingone or several of the components of the flow system.

Depending on the nature of the sample which is to be analysed and otherfactors associated with the sampling and measurement, the preferred flowsystem would not always comprise all the components arranged as shown inFIG. 6, or one or more of the components could be integrated into onecomponent. The present example discusses several possible constructions.

A—A Flow System Contained in a Disposable Unit

Several applications of the present invention can be based on a flowsystem contained in a removable and disposable unit or in a unit whichcan be regenerated. Such a system will have a number of advantages,including the following: Elimination of a stationary flow system thatwould need maintenance such as cleaning. The possibility of being ableto sample and measure without any further handling of a sample, whichmakes the handling of hazardous material more safe.

One such flow system unit 610 is based on following components: An inlet601 where the sample can be introduced in the flow system unit,preferably a valve 603 close to the inlet or integrated with the inletand allowing liquid sample to flow only in one direction, preferably achemical container 604 for any addition of chemical components,preferably a mixing chamber or a manifold 605 allowing the sample andany chemical components to mix, a sample compartment 606 where ameasurement of any signal from the sample will be made, a valve 607controlling the flow of the sample through the sample compartment,preferably a valve which can allow gas or air to pass freely but whichcloses substantially irreversibly upon contact with the sample, andfinally a pump 608 capable of moving the sample from the inlet to orpast the valve 607.

When it is intended that any sample entering the inlet can be retainedwithin the flow system unit upon completion of the analysis, an outletfrom the flow system unit through which the sample could leave thesystem will normally not be provided. Upon completion of the analysis,such a flow system unit can be safely disposed of or regeneratedregardless of the nature of the sample or any chemical components addedto the sample.

B—A Flow System Contained in a Disposable Unit for the Sampling of LargeVolumes Analysed by Multiple Measurements

For some purposes, it may be interesting to be able to measurerelatively large volumes of sample material by multiple measurements ofa number of individual samples taken from a larger volume. This may, forexample, apply when assessing the possible presence and, if present, theconcentration, of bacteria which are objectionable even when beingpresent in very small numbers, such as Salmonella. In such as case, itmay be of interest to perform a small, or a large, series ofmeasurements of “normal volume” samples taken from a larger, butwell-defined, volume of sample material, and then optionally relatingthe results from the small or larger series of volumes to thewell-defined larger volume. According to the present invention, alsothis can be accomplished using a flow system contained in a removableand disposable unit or a unit which can be regenerated. There can beseveral advantages of such a system, including: Improved sensitivity andprecision due to multiple measurements and thereby measurement of alarger total volume. Elimination of a stationary flow system which wouldneed maintenance such as cleaning. The possibility of being able tosample once and then measure several times without any further handlingof a sample makes the handling of hazardous material more safe.

One such flow system unit 610 can be based on following components: Aninlet 601 where the sample can be introduced in the flow system unit,preferably a valve 603 close to the inlet or integrated with the inletand allowing liquid sample to flow only in one direction, preferably achemical container 604 for any addition of chemical components,preferably a mixing chamber or a manifold 605 allowing the sample andany chemical components to mix and having volume at least correspondingto the volume of the large sample with added chemical components, asample compartment 606 of a “normal volume” where measurement of anysignal from the sample is made sequentially on a series of sampleswithdrawn from the large sample, a valve 607 controlling the flow of theindividual sample through the sample compartment, and finally a pump 608which, in connection with the individual measurements, is capable ofpassing at least a portion of the sample contained in the mixing chamberto the sample compartment for the measurement, the pump preferablyhaving capacity to retain, in a large sample entering mode, at least thevolume of sample entering the inlet.

The flow system would need the controlling of at least one valve and/ora pump allowing different portions of the sample to be analysed at atime.

C—A Flow System Contained in a Disposable Unit for the Sampling of LargeVolumes Analysed by a Single Measurement

It is often of interest to be able to measure a large volume of sample.Also this can be accomplished using a flow system contained in aremovable and disposable unit or a unit which can be regenerated. Theadvantage of such system would include: Improved sensitivity andprecision due to measurement of a large volume. Elimination of astationary flow system that would need maintenance such as cleaning. Thepossibility of being able to sample and measure without any furtherhandling of a sample makes the handling of hazardous material more safe.

One such flow system unit 610 could be based on the followingcomponents: An inlet 601 where the sample is introduced in the flowsystem unit, preferably a pump 602 or a valve 603 close to the inlet orintegrated with the inlet and allowing liquid sample only to flow in onedirection, passing the sample to a particle retaining means 605preferably containing means to hold at least the volume of sampleentering the inlet, or connected to an outlet 609 allowing the sample toleave the flow system unit, preferably a chemical container 604 for anyaddition of chemical components connected to the particle retainingmeans, preferably a mixing chamber of manifold 605 allowing the sampleand any chemical components to mix, a sample compartment 606 where ameasurement of any signal from the sample would be made, a valve 607controlling the flow of the sample through the sample compartment,preferably a valve which can allow gas or air to pass freely but closessubstantially irreversibly upon contact with the sample, and finally apump 608 capable of passing at least a portion of the sample containedin the particle retaining means through the chemical component containerto the sample compartment for the measurement.

With slight variation in the arrangement of the components it would bepossible to measure the signal from the particles in the sample whilestill retained on or in the particle retaining means. One possiblearrangement could be to include the particle retaining means in thesample unit, and passing the sample through the sample unit. Thenpreferably to pass any chemical component through or into the samplecompartment to allow the mixing with any retained particle and finallyto perform the measurement.

D—A Stationary Flow System for the Measurement of Several Samples

In many applications it would be of interest to be able to measure morethan one sample without the replacement of any part of the flow systembetween analysis. Such a flow system would normally be a stationary partof an analytical instrument.

One such flow system could be constructed as follows: an inlet 601 wherethe sample enters the flow system and a pump 602 for the flowing of thesample, preferably a valve 603 for controlling the flow, preferably areservoir for chemical components 604 which can preferably containchemical components for the measurement of more than one sample,preferably a mixing chamber 605 for the mixing of the sample and anychemical component, a sample compartment 606 for the measurement of asignal from the sample, preferably a valve 607 controlling the flow ofsample through the sample compartment, and an outlet 609 where thesample leaves the flow system.

EXAMPLE 5 A Disposable Measurement and Sampling Unit for the Assessmentof the Number of Somatic Cells in a Volume of Milk

The components of a flow system which can be used for the assessment ofsomatic cells in milk are shown in FIG. 7:

An inlet 701 where the sample is introduced to the flow system, acompartment 702 containing reagents prior to analysis, a compartment 703which allows a substantially homogeneous mixing of the milk with thereagents, a sample compartment 704, a valve 705 controlling the flowfrom the sample compartment, a piston pump capable of producing vacuum,consisting of a chamber 706 with connection to the flow system and theexterior and a piston 707 which has such dimensions that it fits closelyin the chamber, thus resulting in a low pressure on the flow system sideof the pump chamber when moved into the chamber.

Prior to analysis, the sample inlet is immersed in the milk sample to beanalysed. While the sample inlet is immersed in the milk sample, thesample is introduced to the flow system of the disposable measurementand sampling unit by moving the piston at least partially into the pumpchamber. The vacuum produced should be of such magnitude that the milksample flows through the reagent compartment, thus dissolving orsuspending at least a portion of the reagents present in thecompartment, and into the mixing compartment.

Preferably, the mixing compartment has a sufficiently large volume tosecure that it becomes only partially filled with the milk and anyreagents dissolved or suspended, thus allowing the content of the mixingcompartment chamber to flow freely in the chamber and thus to beeffectively mixed.

After the mixing has been completed, the piston is moved further intothe pump chamber, thus producing vacuum capable of passing the milksample into the sample compartment and further into the valve whichcloses upon contact with the sample thus substantially stopping the flowof sample through the sample compartment.

The dimension of the reagent compartment should be adequate to allow thestorage of the reagents used, for instance 2 mg Triton X100(t-Octylphenoxypolyethoxyethanol) and 5 μg Propidium Iodide(CAS-25535-16-4). The shape of the void inside of the reagentcompartment should preferably be such as to enhance the solvation orsuspension of the reagents contained in the compartment prior toanalysis.

The mixing compartment has a volume of about 200 μl, depending on thetotal amount of milk used for the analysis. The shape of the void of themixing compartment should be such as to allow any liquid to flow fromone boundary to another thus allowing a thorough mixing.

The sample compartment consists of two substantially parallel planesforming a void with the approximate dimensions of 10×10×0.07 mm (height,with, depth). Depending on the method used for the production of theunit, then either the average depth of the sample compartment issubstantially identical for all individual disposable measurement andsampling units thus allowing reproducible volumes of milk to be presentin the sample compartment during analysis or it is possible to labeleach individual disposable measurement and sampling unit, this labelidentifying the approximate depth of the sample compartment thusallowing the instrument to compensate the assessment of somatic cells inmilk for the varying depth of the sample compartment.

The valve used in the disposable measurement and sampling unit is onewhich is capable of letting air pass through until a liquid comes incontact with it. When a liquid has been in contact with the valve it issubstantially irreversibly closed thus allowing neither liquid nor airto pass through it. One such valve can be constructed by using fibrematerial from Porex Technologies GmbH, German (XM-1378, EDP#NS-7002).

EXAMPLE 6 An Instrument for the Assessment of the Number of SomaticCells in a Volume of Milk

FIG. 8 illustrates an instrument which can be used for the assessment ofthe number of somatic cells in a volume of milk sample. The instrumentis powered by either an external power source 801 or by an internalpower source such as a lead acid (12V 2.2 Ah) rechargeable battery 803,manufactured by Wetronic Inc. (WE12-2.2).

The Power supply/battery charger 802 supplies the different units of theinstrument. The power supply can use power from either the external orthe internal power source, and is capable of switching between the twosources during operation. It is possible to reduce the power consumptionwhen the instrument is in stand-by.

The assessment of the number of somatic cells is performed by detectinga fluorescence signal originating from a fluorochrome bounded to DNAwithin somatic cells present in the sample compartment 807. The samplecompartment is defined by two substantially parallel planes oftransmitting material thus forming a compartment with dimensions ofabout 10×10×0.7 mm (height, width, depth).

The fluorescence is generated by passing light of high energy(excitation light of wavelength 550 nm or less) through the samplecompartment, with direction towards the detection module 811. The source804 of the excitation light can be either a halogen lamp of typeOSRAM-64255 (8V, 20 W Photo Optic Lamp) or a number of light emittingdiodes, for instance 4 or more, of type NSPG-500S or NSPE-590S (NichiaChemical Industries Ltd., Japan).

In order to remove substantially any component from the excitation lightwith wavelength above 550 nm from reaching the sample compartment, anoptical filter 805 is inserted in the light path. This filter of thetype Ferroperm SWP550, double sided interference filter on a 2 mmsubstrate (Hoya, CM-500) which absorbs infra-red radiation.

To further preventing infra-red radiation from reaching the samplecompartment a heat absorbing filter 806 is placed in the light path.This filter is of the type Schott KG5 or KG3 (3 mm in thickness). Thisfilter can be omitted if light emitting diodes are used as light source.

The light emitted from the sample compartment is focused onto thesensors of the detection module by the use of at least one lens 808.This lens is a standard ×4 microscope objective with numerical apertureof 0.10 (Supplied by G. J. Carl Hansens Eftf., Denmark). The lens isarranged in such a way as to give an image of an object in the samplecompartment on the sensors of the detection module which hasapproximately the same size as the original object (magnificationapproximately ×1).

In order to remove substantially any component from the light emittingfrom the sample compartment with wavelength below 575 nm from reachingthe detection module, an optical filter 809 is inserted in the lightpath. This filter is of the type Schott OG590 (thickness 3 mm).

To further prevent infra-red radiation from reaching the detectionsystem a heat absorbing filter 810 is placed in the light path. Thisfilter is of the type Schott KG5 or KG3 (3 mm in thickness). This filtercan be omitted if light emitting diodes are used as light source.

The filtered light from the sample compartment is detected by a chargecouple device (CCD) 811 of the type GCA325 KBL (supplied by L&GSemicon). The CCD is equipped with 510×492 detection elements.

The electrical information from the CCD is amplified and measured by ananalogue to digital converter module 812 (ADC).

The operation of the instrument is controlled by the computer unit 813.The computer is a Motorola DSP56824 16 bit digital signal processor,equipped with non-volatile storage capacity for long time storage(EEPROM) as well as volatile storage capacity (RAM). The computergathers information about the measured light intensity of each detectionelement of the CCD from the ADC module and uses it for the assessment ofthe number of somatic cells in the milk sample present in the samplecompartment. The computer module is equipped with a real time clock.

The result of the assessment of the number of somatic cells in the milksample is presented on a display 815 of type MDLS16166-3V (supplied byVaritronix).

The result of the assessment of the number of somatic cells in the milksample can also be transmitted to an external computer (not shown) bythe use of the output port 816.

The user of the instrument can control its operation, and enter relevantinformation through a collection of keys forming a key-pad 814. Thekey-pad is a 16 keys module of type ECO 16250 06 SP.

EXAMPLE 7 An Instrument for the Assessment of the Number of Bacteria ina Volume of Aqueous Sample

FIG. 8 illustrates an instrument which can be used for the assessment ofthe number of bacteria in a volume of aqueous sample. The instrument ispowered by either an external power source 801 or by an internal powersource such as a lead acid (12V 2.2 Ah) rechargeable battery 803,manufactured by Wetronic Inc. (WE12-2.2).

The Power supply/battery charger 802 supplies the different units of theinstrument. The power supply can use power from either the external orthe internal power source, and is capable of switching between the twosources during operation. It is possible to reduce the power consumptionwhen the instrument is in stand-by.

The assessment of the number of somatic cells is performed by detectinga fluorescence signal originating from a fluorochrome bounded to DNAwithin somatic cells present in the sample compartment 807. The samplecompartment is defined by two substantially parallel planes oftransmitting material thus forming a compartment with dimensions ofabout 10×10×0.7 mm (height, width, depth).

The fluorescence is generated by passing light of high energy(excitation light of wavelength 550 nm or less) through the samplecompartment, with direction towards the detection module 811. The source804 of the excitation light can be either a halogen lamp of typeOSRAM-64255 (8V, 20 W Photo Optic Lamp) or a number of light emittingdiodes, for instance 4 or more, of type NSPG-500S or NSPE-590S (NichiaChemical Industries Ltd., Japan).

In order to remove substantially any component from the excitation lightwith wavelength above 550 nm from reaching the sample compartment, anoptical filter 805 is inserted in the light path. This filter of thetype Ferroperm SWP550, double sided interference filter on a 2 mmsubstrate (Hoya, CM-500) which absorbs infra-red radiation.

To further preventing infra-red radiation from reaching the samplecompartment a heat absorbing filter 806 is placed in the light path.This filter is of the type Schott KG5 or KG3 (3 mm in thickness). Thisfilter can be omitted if light emitting diodes are used as light source.

The light emitted from the sample compartment is focused onto thesensors of the detection module by the use of a lens 808. This lens canbe a standard ×4 microscope objective with numerical aperture of 0.10(Supplied by G. J. Carl Hansens Eftf., Denmark) or a standard ×10microscope objective. The lens is arranged in such a way as to give animage of an object in the sample compartment on the sensors of thedetection module which has approximately four to six times larger thanthe original object (magnification approximately between ×4 and ×6).

In order to remove substantially any component from the light emittingfrom the sample compartment with wavelength below 575 nm from reachingthe detection module, an optical filter 809 is inserted in the lightpath. This filter of the type Schott OG590 (thickness 3 mm).

To further remove infra-red radiation from reaching the detection systema heat absorbing filter 810 is placed in the light path. This filter isof the type Schott KG5 or KG3 (3 mm in thickness). This filter can beomitted if light emitting diodes are used as light source.

The filtered light from the sample compartment is detected by a chargecouple device (CCD) 811 of the type GCA325 KBL (supplied by L&GSemicon). The CCD is equipped with 510×492 detection elements.

The electrical information from the CCD is amplified and measured by ananalogue to digital converter module 812 (ADC).

The operation of the instrument is controlled by the computer unit 813.The computer is a Motorola DSP56824 16 bit digital signal processor,equipped with non-volatile storage capacity for long time storage(EEPROM) as well as volatile storage capacity (RAM). The computergathers information about the measured light intensity of each detectionelement of the CCD from the ADC module and uses it for the assessment ofthe number of bacteria in a volume of aqueous sample present in thesample compartment. The computer module is equipped with a real timeclock.

The result of the assessment of the number of bacteria in a volume ofaqueous sample is presented on a display 815 of type MDLS 16166-3V(supplied by Varitronix).

The result of the assessment of the number of bacteria in a volume ofaqueous sample can also be transmitted to an external computer (notshown) by the use of the output port 816.

The user of the instrument can control its operation, and enter relevantinformation through a collection of keys forming a key-pad 814. Thekey-pad is a 16 keys module of type ECO 16250 06 SP.

EXAMPLE 8 The Assessment of the Number of Somatic Cells in a Volume ofMilk According to the Present Invention Compared to the Results of aRoutine Instrument

The result of the assessment of the number of somatic cells in a volumeof milk according the present invention was compared to the resultsobtained from a FossoMatic 400 routine instrument (Foss Electric,Denmark).

191 milk samples from individual cows, were measured on the FossoMaticinstrument according to the instructions provided by the producers (FossElectric, Denmark).

Upon the completion of the measurement on the FossoMatic a 1 ml (±2%)portion of the remaining sample was taken and mixed with 1 ml (±1%) ofaqueous reagent solution, resulting in a milk solution containing 0.25%(w/v) Triton X-100 (t-Octylphenoxypolyethoxyethanol) and 25 μg/mlpropidium iodide (CAS-25535-16-4).

The assessment of the number of somatic cells in a volume of milk wasperformed on an instrument according to the present invention, equippedwith an excitation module comprising a halogen light source, OSRAM-64255(8V, 20 W Photo Optic Lamp), an optical filter, Ferroperm SWP550 (doublesided interference filter on a 2 mm substrate (Hoye, BG-39) whichabsorbs infra-red radiation) and a heat absorbing filter, (Schott KG5, 3mm in thickness), and a detection module comprising a focusing lens,standard ×4 microscope objective with numerical aperture of 0.10,arranged in such a way as to give a magnification of approximately ×10on the sensor elements, an optical filter, (Schott OG590, thickness 3mm), and a heat absorbing filter, Schott KG5 (3 mm in thickness), and aCCD detector, SONY-CX 045 BL.

A portion of the milk solution was placed between two substantiallyparallel plates of glass, placed approximately in the focus plane of thedetection module, and irradiated by excitation light emitted from theexcitation module. The distance between the two parallel glass plateswas approximately 100 μm. The volume being detected by the detectionmodule, defined by the size of the CCD, the magnification used, and thedistance between the parallel glass plates was equivalent toapproximately 1 μl, thus containing approximately 0.5 μl of milk.

The sample compartment was a stationary flow cell. The milk solution wasplaced into the sample compartment by the use of a peristaltic pump,situated down-stream from the sample compartment. In order to reduce themovement of the sample inside the flow cuvette a valve was placed in theflow system, immediately adjacent to the sample compartment.

Each observation was based on the measurement of two portions of themilk solution. The two measurement were treated in such manner that thesecond measurement was numerically subtracted from the first measurementdiscarding all values having negative result by assigning zero thesevalues. The number of somatic cells represented in each observation wasdetermined by identifying and counting the number of “peaks” in theresulting observation.

The assessment of the number of somatic cells in a volume of milk waspresented as the number of counted peaks in two observations from thesame sample solution, thus presented as the number of somatic cells per1 μl of milk.

The results obtained by the two methods are given in FIG. 9 as a graphof the assessment according to the present invention (labelled “CellCounter”) vs. results obtained by the FossoMatic instrument. FIG. 9Ashows the graph of the result of the 191 samples. FIG. 9B shows theresult obtained when considering those samples having an estimatednumber of somatic cells of less than 400 cells/μl.

Conclusion

The conclusion from the test described above as shown in FIG. 9 is thatthe assessment of the number of somatic cells in milk according to thepresent invention is generally in good agreement to the results obtainedby the FossoMatic instrument.

EXAMPLE 9 The Effect of the Concentration of Fluorochrome on theAssessment of Biological Particles in a Scattering Biological SampleMaterial According to the Present Invention

When performing the assessment of biological particles in biologicalsample material according to the present invention it is often ofinterest to maximise the volume of the sample solution being analysed.This can for instance be accomplished by increasing the depth of thesample being analysed.

Apart from any limitation by effective focusing depth can imply thescattering or attenuating property of the sample being analysed canlimit the effective depth of the sample.

When analysing samples containing a high number of particles or otherconstituents being capable of causing scattering or other attenuation ofany signal being measured this can limit the effective depth of thesample. The cause of this can be that any signal originating from aparticle situated relatively deep in the sample is attenuated whiletrawling towards the boundaries of the sample.

One such sample material is milk. Milk contains both a high number offat globules and protein micelles. As a result of this milk is a highlyscattering media.

When assessing the number of somatic cells in a volume of milk by theuse of a method based on the measurement of a fluorescence signal fromthe sample the scattering properties of the milk can limit the effectivedepth of the sample being analysed. This is partly due to theattenuation of any signal originating from cells situated deep in thesample but also due to the fact that the variations in the backgroundsignal, caused by any fluorochrome molecules on free form, increases,thus making it more difficult to identify the signals originating fromthe somatic cells.

The present example illustrates the effect of the concentration of thefluorochrome on the number of somatic cells which can be identified in amilk sample. The sample was a single cow sample, preserved withbronopol. The estimated number of somatic cells in the sample suggestedthat between 1150 and 1200 objects should be counted under the presentconditions.

The sample was measured in a measuring cell consisting of twosubstantially parallel glass plates, separated by a distance of about100 μm, representing the depth of the sample. Two measurement ofdifferent portions of the sample were taken and the resulting image wasconstructed by subtracting the later measurement from the firstmeasurement and then multiplying those results which were negative bythe value −1 thus producing a final image consisting entirely of valueslarger than or equal to 0.

Reagents were added to the milk sample, amounting to about 5% of thevolume of the sample being analysed (resulting in an effective thicknessof the milk equivalent to about 95 μm). The reagents contained TritonX100 (t-Octylphenoxypolyethoxyethanol) resulting in a finalconcentration of 0.24% (w/v) and Propidium Iodide (CAS-25535-16-4) asfluorochrome resulting in final concentrations ranging between 89 and2.4 μg/ml.

To compensate for the reduced signal due to the decreasing concentrationof Propidium Iodide the electronical gain of the detection system wasadjusted accordingly.

The result of the experiment is given in FIG. 10 which shows a graph ofthe number of objects which were counted vs. the concentration of thePropidium Iodide.

Conclusion

The conclusion from the investigation as described above is that whenmeasuring samples having scattering properties, it can be possible toextend the depth of the sample being analysed by reducing theconcentration of the fluorochrome.

EXAMPLE 10 Processing of a Two Dimensional Image

The result of a measurement of signals from particles by an array ofdetection elements, such as a charged coupled deceive (CCD), forinstance SONY-CX 045 BL, according to the present invention, can bevisualised as a two dimensional image where the intensity of eachdetection element can be represented by a density or a colour.

FIG. 11A gives a presentation of a segment of an image from ameasurement of somatic cells in milk. The intensities on which FIG. 11Ais based is given in Table 1 below. In FIG. 11 the intensity isrepresented by shades of grey, such that low intensity has a lightershade and high intensity has a darker shade.

Assuming that each somatic cell gives rise to an image which has thesize of approximately 2×2 detection elements we can estimate the numberof somatic cells being represented in FIG. 11A to be approximately 10.

The task of having a computer determining the number of particles beingrepresented in a measurement involves the construction of a set of rulesor instructions for the computer, which when applied to the result of ameasurement gives an estimate of the number objects.

One such simple rule could be to identify the number of detectionelements which have intensity above a given threshold value. Assumingthat each object on average is represented by an intensity in a givennumber of detection elements the number of identified detection elementcan be adjusted to give an estimate of the number of objects. Such amethod is dependent on that an approximately correct estimate of thesize of the image of an object is available.

In the following a pre-processing of the image is presented which makesthe previous method of assessment less dependent on the size of an imageof an object. The effect of the processing is to concentrate theintensity information in a given region of the image to substantiallyone number.

a) The first step of the processing is to define a region which has asize which is at least the same as the size of the image of an objectwhich is to be detected. In the present example this region is of thesize 5×5 detection elements but regions of different size can be useddepending on the nature of the image of the object being analysed. Thisregion is placed in the two dimensional co-ordinate system defined bythe detection elements. In this example this region is initially placedin the upper left corner of the co-ordinate system. For each of suchregion a data value is defined which will hold the value representingthe region.

The next step is to adjust the value of the data element representingthe region. This is done by firstly by considering the intensitygradient around the centre of the region and secondly by considering theintensities of the detection elements positioned adjacent to the centreof the region. It is possible to interchange the order of these steps.This produces different result, depending on the order chosen.

b) The investigation of the gradient around the centre of the region isbased on investigating the intensity values of at least two detectionelements. In the present example we estimate the intensity values ofthree detection elements including the detection element situated in thecentre of the region. A total of 8 gradients originating at the centreof the region and with a direction horizontal, vertical or diagonalrelative to the centre of the region.

Assuming the identification of detection elements as follows, where thecentre of the region is identified as A: 1 2 3 4 5 1 I — B — C 2 — I_(o)B_(o) C_(o) — 3 H H_(o) A D_(o) D 4 — G_(o) F_(o) E_(o) — 5 G — F — E

The eight different gradients are defined by the following detectionelements: [A,B_(o),B], [A,C_(o),C], [A,D_(o),D], [A,E_(o),E],[A,F_(o),F], [A,G_(o),G], [A,H_(o),H], [A,I_(o),I].

The value representing the region can be adjusted in different waydepending on the result of the gradient testing. In the present examplethe value is adjusted to zero if one of the following gradient test istrue, defined as following:

The value of the region is zero if(

-   -   (A<B AND A<B_(o)) OR (A<C AND A<C_(o)) OR (A<D AND A<D_(o)) OR    -   (A<E AND A<E_(o)) OR (A<=F AND A<F_(o)) OR (A<=G AND A<G_(o))        OR)    -   (A<=H AND A<H_(o)) OR (A<I AND A<I_(o))) is true

The value of the region can either be stored separately for the regionor it can be used to replace the value of the detection elementidentified as A. In the present example the value of the region is usedto replace the value of A and will be used as intensity value of thedetection element in subsequent analysis.

When the previous step has been completed a new range according to a) isdefined. The new range is placed on a different position of the image.In the present example the position of the new range is defined bymoving the range down a column by two rows. When the end of the columnhas been reached the next range is position by moving to the first rowand two columns to the right. The range is moved in this way untilsubstantially the entire image has been investigated.

c) When substantially the entire image has been investigated accordingto b), the second step in defining a value representing the regioninvolves adjusting the value of each range based on the detectionelements which are situated immediately adjacent to the centre value ofthe region. In the present example this is done by assigning the resultof the following expressions to the value of each range:

A=if(A>=B & A>=H AND A>=I & A>=C & A>=D) then max(A,B_(o)) else A)

A=if(A>=B & A>=C & A>=D) then max(A,C_(o)) else A)

A=if(A>=D & A>=B & A>=C & A>=E & A>=F) then max(A,D_(o)) else A)

A=if(A>D & A>E & A>F) then max(A,E_(o)) else A)

A=if(A>=F & A>D & A>E & A>G & A>H) then max(A,F_(o)) else A)

A=if(A>F & A>G & A>H) then max(A,G_(o)) else A)

A=if(A>=H & A>F & A>G & A>I & A>B) then max(A,H_(o)) else A)

A=if(A>H & A>I & A>G) then max(A,I_(o)) else A)

Value=A

(“&” represents the logical operation AND)

When the previous step has been completed a new range is definedpreferably in the same manner as was done previously.

The results of processing the data in Table 1 and FIG. 11A according tothe method outlined above is given in Table 2 below and in FIG. 11B.

When both steps b) and c) have been completed for substantially theentire range of detection elements the estimation of the number ofobjects being represented in the image can be done on the bases of thevalues estimated for each range.

The image of each object which signal is represented in a range ofdetection elements which is of comparable size to the ranges being usedfor the processing, or smaller, will substantially result in only onevalue when both steps b) and c) have been completed for substantiallythe entire range of detection elements. This makes it possible toestimate the total number of objects represented in an image bycomparing the value of each of the ranges to give a threshold valuesince each object is substantially only represented in one value. TABLE1 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1920 21 22 1 1 8 4 1 5 11 2 2 5 4 2 9 17 12 3 6 14 2 5 2 2 1 2 8 12 5 6 45 4 4 10 10 8 9 4 7 9 5 3 9 6 2 8 2 1 3 5 6 3 5 2 8 12 9 2 16 3 12 2 6 58 9 2 8 3 5 4 4 3 3 4 4 8 5 8 8 4 7 3 7 9 8 11 2 5 7 5 6 2 1 2 5 6 3 5 721 15 8 6 5 3 9 9 49 29 20 3 7 3 3 7 9 2 6 5 2 4 16 171 179 22 2 6 6 734 71 132 53 12 10 3 3 2 12 13 3 7 6 3 11 17 81 109 31 14 14 2 15 20 4138 25 10 3 7 6 5 12 9 8 3 6 11 4 10 18 7 12 3 6 10 14 5 4 14 4 7 2 13 28 13 4 9 4 2 12 8 2 6 9 9 5 5 5 3 11 9 12 9 20 88 165 22 3 2 10 8 2 2 58 7 2 4 6 2 6 7 5 2 14 8 30 167 171 53 7 9 5 11 5 4 3 10 1 1 2 5 8 6 3 62 7 10 23 30 31 18 10 7 5 12 4 5 5 1 3 6 7 2 1 4 2 15 4 10 43 169 25 105 6 11 6 6 13 7 1 4 7 4 8 5 8 11 14 7 5 4 5 12 33 14 3 11 3 2 9 14 3 7 23 5 13 8 3 2 7 2 10 4 2 6 5 5 4 10 1 22 39 7 15 8 3 4 7 2 2 2 4 5 4 3 411 2 7 4 2 6 2 15 74 162 16 4 2 14 2 2 7 2 3 7 3 1 5 2 1 5 4 1 2 2 15 4060 8 17 2 6 5 4 3 9 5 3 8 2 5 5 4 3 8 5 12 3 3 6 1 4 18 2 2 9 8 2 11 910 9 1 2 3 3 3 2 5 7 2 12 11 19 37 9 19 1 6 3 2 7 2 3 8 10 2 11 5 3 12 75 2 3 7 18 30 69 20 10 6 2 2 5 6 1 7 4 5 7 1 1 7 2 5 8 8 8 11 7 30 21 52 2 3 13 4 4 11 4 4 5 5 4 8 4 3 3 2 4 4 1 11 11 12 13 14 15 16 17 18 1923 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 1 4 11 3 3 5 1613 5 1 8 2 7 1 6 7 3 2 5 2 2 3 7 4 2 8 57 172 33 2 10 2 10 11 13 9 6 1 58 1 3 6 7 4 3 7 98 166 53 6 4 9 8 30 16 11 9 2 1 6 4 8 4 7 6 8 20 20 6 34 7 25 171 175 29 5 2 1 4 2 5 5 3 8 5 3 4 9 1 6 9 13 24 69 63 20 7 3 2 86 5 2 4 9 3 2 10 3 3 5 13 12 14 6 6 5 5 8 1 3 7 4 2 5 1 2 6 2 5 7 1 9 26 3 2 4 2 10 3 8 2 11 10 2 6 14 4 6 3 5 4 4 5 2 5 2 6 3 3 4 9 5 9 3 8 48 5 4 1 6 8 10 2 6 6 2 3 1 6 10 2 4 6 6 2 2 3 10 4 2 4 11 21 88 44 9 2 33 5 11 9 3 9 8 3 1 3 1 4 5 4 6 68 164 164 19 2 4 2 12 1 11 3 4 2 8 3 4 94 4 5 33 123 54 4 2 5 1 6 13 5 9 8 1 1 8 7 9 3 3 1 11 11 38 17 5 3 4 514 35 9 4 5 4 4 6 6 13 2 5 11 21 41 11 4 7 5 3 7 15 125 20 7 6 6 4 10 710 2 1 6 7 10 12 5 5 7 12 16 44 10 36 169 40 3 13 8 11 4 7 3 3 3 2 2 3 713 8 17 3 7 31 87 25 3 4 3 3 3 4 5 1 2 6 1 7 6 2 18 57 25 23 1 11 3 1 1011 8 12 4 8 14 6 4 3 4 4 9 19 91 25 16 2 7 7 3 2 9 6 1 3 2 4 7 1 5 9 1220 42 8 9 6 11 1 2 4 6 2 4 6 3 4 2 8 10 6 2 21 13 10 3 3 2 3 4 5 6 2 6 52 8 2 9 3 4 3

TABLE 2 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19 20 21 22 1 2 1 3 0 0 12 0 0 0 0 9 0 6 4 2 5 4 0 0 7 0 132 0 0 0 06 3 7 0 179 14 6 0 0 12 10 0 13 8 4 9 12 0 12 6 14 9 14 0 171 0 10 5 110 7 0 8 7 7 23 0 0 0 12 6 13 7 8 0 14 15 10 169 0 11 0 14 7 15 7 13 0 00 11 7 0 0 0 16 8 17 2 7 3 0 5 0 8 12 15 0 18 9 19 0 11 0 10 11 0 7 0 110 20 21 11 12 13 14 15 16 17 18 19 23 24 25 26 27 28 29 30 31 32 33 3435 36 37 38 39 40 41 1 2 1 3 7 0 0 172 0 0 0 0 0 4 2 5 0 9 0 0 4 0 175 70 6 3 7 0 11 6 0 7 0 0 5 0 8 4 9 0 0 14 0 0 10 5 5 0 10 5 11 9 9 2 10 511 0 164 0 12 6 13 0 0 0 8 4 11 0 0 0 14 7 15 162 5 0 10 13 11 0 41 0 168 17 0 169 0 0 0 5 0 0 7 18 9 19 91 0 3 0 11 4 0 14 0 20 21

EXAMPLE 11 Assessment of the Number of Bacteria in an Aqueous Sample

Using the instrument described in example 7 the number of bacteria in anaqueous sample was assessed according to the present invention. Theexperiment was conducted as follows:

Glucose, in the amount of 0.3 g, was added to a portion of 30 ml ofBuffered Peptone Water (LAB03627, Bie & Berntsen, Denmark). This brothwas inoculated with 100 μl of a raw milk sample containing a naturalbacterial microflora. After inoculation the broth was incubated for 24hours at approximately 30° C.

Following incubation the broth was strongly turbid. Empirically such aculture contains from 1×109 to 2×109 colony forming units per ml.

A 25 μl portion of the incubated broth was transferred to 1 ml of anaqueous reagent solution containing 0.13% Triton X-100, 13 μg/mlpropidium iodide and 0.5% ethylenediaminetetraacetic acid (EDTA, Sigma E4884).

After mixing a portion of the broth/reagent solution was placed betweentwo substantially parallel glass plates, placed approximately in thefocus plane of the detection module, and irradiated by excitation lightemitted from the excitation module.

The distance between the two parallel glass plates was approximately 100μm. The volume being detected by the detection module, defined by thesize of the CCD, the magnification used, and the distance between theparallel glass plates was equivalent to approximately 0.04 μl, thuscontaining approximately 0.001 μl of the incubated broth.

Two measurements of different portions of the sample were taken. Theresult from the second measurement was subtracted from the firstmeasurement, transforming those values which are below zero to zero bythe formula: Signal_((ij))=MAX((meas1_((ij))−meas2_((ij))),0).

Results

The result from the subtraction of the intensities from the twomeasurements as described above, is represented in FIG. 12. The resultfrom the subtraction of intensities are presented by converting theresulting value for each detection element to the shade of grey, thelight shads representing low intensities and the dark shads representinghigh intensities.

The number of objects represented in the measurement was countedaccording to a method of the present invention, as described in Example10 resulted in an assessment of the number of bacteria in the samplevolume was 1601.

The number of counts in one observation was expected to be in the rangeof 1000 to 2000. Thus it is reasonable to conclude that the instrumentdescribed in example 7 is able to assess the number of bacteria in an aaqueous sample.

DETAILED DESCRIPTION OF EMBODIMENTS

While a number of preferred embodiments have been described above, thepresent invention can be performed and exploited in a large number ofways. In the following, a discussion of a number of measures and detailsrelevant to the invention is given, comprising both preferredembodiments and embodiments which illustrate possibilities of workingthe invention. Some of the embodiments are given as numbered items, tobe understood as brief indications of possible and preferred embodimentsin the light of the remaining claims and description herein.

Detection Elements

In the method of the present invention, the assessment of biologicalparticles in a volume of liquid sample material is made by arranging asample of the liquid sample material in a sample compartment having awall defining an exposing area, transparent to electromagnetic signalsemitted from the sample being exposed to the exterior, and forming animage of electromagnetic signals from the sample in the samplecompartment on an array of detection elements, and processing the imageformed on the array of detection elements in such a manner that signalsfrom the biological particles are identified as distinct from the samplebackground, and based on the signals from the biological particlesidentified assessing biological particles in a volume of liquid samplematerial.

In the present specification and claims, the term “biological particle”designates a particle originating from, or found in living matter, suchas somatic cells, red blood cells, blood platelets, bacteria, yeastcells, fragments of cells, lipid globules, protein micelles, plankton,algae or fraction thereof.

In the present specification and claims, the term “biological samplematerial” designates a liquid sample material of, often biologicalorigin, or material where biological particles might be found, such as:specimen of human origin, specimen of animal origin, drinking water,waste water, process water, sea water, lake water, river water, groundwater, food, feed or components of food and feed, milk or a milkproduct, blood or a blood product, urine, faeces, saliva, specimen froman inflammation, specimen from the petrochemical industry, specimen fromthe pharmaceutical industry, specimen from the food or feed industry, orproduct thereof.

The method allows a sample of the sample material to be analysed whenpractically all components in the sample material are present in thesample during the measurement on which the assessment is based. This isoften practical when the liquid sample material is a biological samplematerial, since it is often associated with considerable difficulties toselectively remove one, or several, or substantially every componentfrom a sample of sample material prior to analysis.

Detection Array

The array of detection elements can be arranged in such a way that theyform a substantially straight line. When using a high number ofdetection elements they can be arranged in two directions in such a waythat the detection elements form a series of substantially parallelstraight lines, and often the array of detection elements is arranged inone plane. This plane of detection elements is often arranged parallelto an inner boundary of the sample compartment.

Signal Conditioning—Hardware

The signal detected by the detection elements is normally anelectromagnetic radiation and it is therefore preferable to have methodsto transform those signals to measurable signal, such as voltage, orelectrical current. Many such signals have a varying background signal,or bias, and it is therefore preferable to have methods which at leastpartly can eliminate those effects. This can often be accomplished byusing a signal in one or several neighbouring detection elements asreference.

Another useful method is one where any varying intensity of thedetection elements is adjusted, preferably by the use of results fromone or several of previous measurements.

Arrays of detection elements are often made up by a high number ofdetection elements, and it can therefore be advantageous to reduce thenumber of measured signals prior to assessment, preferably without theloss of any significant information. One such method is to combine thesignal from one or more detection element to one signal, for instance bycombining 2, maybe more than 2 and even as many as 8 or 16 or 32 or moreinto one signal.

In some situations e.g. in an analog-to-digital conversion it could alsobe of interest to adjust the level of 2, preferably 3, more preferably4, more preferably 5, more preferably 6, more preferably 7, morepreferably 8, more preferably more than 8, separate output channels insuch a way that one, preferably more than one, of the output channelshas/have substantially different level from the other output channel(s),where the identification of which of the output channels, or combinationthereof, has substantially different output level, is correlated to theintensity of said signal.

For the analysis of any measured signal it is often necessary todigitalise the signal, in such a way that a given intensity of anysignal is transformed into a digital representation. This can be done byhaving a series of channels, were the information about which of thesechannels has signal which differs from the other channels determines theintensity, or even by having more than one of this channels forming acombination, preferably in a way similar to binary representation.

Focusing—Lenses

Signals from at least a portion of the sample are focused onto the arrayof detection elements, by the use of a focusing mean, preferably by theuse of one lens, more preferably by the use of two lenses, morepreferably by the use of more than two lenses. The number of lenses usedfor the focusing system can affect the complexity of any measuringsystem. A system with two or more lenses is normally preferred while asystem with only two or even only one lens is preferable.

Adaptive Focusing

The focusing of a signal from the sample onto any detector is dependenton the position of the sample relative to any detector. When theconstruction of measuring system is such, that the relative position ofthe sample and any detector can vary, then there is advantage in beingable to adjust the focusing of the system. This can often be achieved byfirst taking at least one measurement of any signal from the sample andthen on the bases of is, to adjust the focusing of the system. Thisprocedure can be repeated a number of times in order to obtainacceptable focusing. In the same manner the focusing of signal from thesample or sample material is adjusted, preferably where the extend ofthe adjustment is determined by at least one measurement of a signalfrom the sample.

Focusing—Enlargement

In order to increase the amount of electromagnetic radiation which isdetected by a detection element, it is often preferable to use one ormore lenses to focus the signal from the sample onto the array ofdetection elements. The magnification of such focusing can be differentfrom 1/1, depending on the set-up of other components of the system, orthe particles or sample material used. For instance can enlargement bepractical when assessing morphological properties of a particle.

In situations where the particles are relatively small the ratio of thesize of a biological particle, to the size of the image of thebiological particle on the array of detection elements could be 1/1 orless, preferably less than 1/1 and higher than 1/100, and even less than1/1 and higher than 1/40, or in other preferred situations less than 1/1and higher than 1/10, and even in some situations it is preferred theratio being less than 1/1 and higher than ¼, more preferably less than1/1 and higher than ½.

Focusing— 1/1

When the particles in question have dimensions which is comparable tothe size of a detection element, it is often preferred to havemagnification of about 1/1, thus focusing the image of any particle onany one or just few detection elements. This can under some conditiongive favourable detection of any signal.

In these situations it is preferred that the ratio of the size of abiological particle, to the size of the image of the biological particleon the array of detection elements is in the interval between 5/10 and20/10, preferably in the interval between 6/10 and 18/10, morepreferably in the interval between 7/10 and 16/10, more preferably inthe interval between 8/10 and 14/10, more preferably in the intervalbetween 9/10 and 12/10, more preferably substantially equal to 10/10.

Focusing—Reduction

When analysing particles which have dimensions which are comparable to,or bigger than the detection elements used, it is often advantageous toreduce the size of the image of such particle, to a degree where thesize of the image is comparable to the size of a detection element.

In these situations it is preferred that the ratio of the size of abiological particle, to the size of the image of the biological particleon the array of detection elements is 1/1 or less, preferably less than1/1 and higher than 1/100, more preferably less than 1/1 and higher than1/40, more preferably less than 1/1 and higher than 1/10, morepreferably less than 1/1 and higher than ¼, more preferably less than1/1 and higher than ½.

Focusing—Aspect Ratio

Surprisingly it was found that the aspect ratio of an image can beconsiderably distorted on the array of detection elements, without thathaving considerable negative effect on the assessment of particles. Insuch a situation it preferred that the ratio of the shorter to thelonger of the two dimensions of the image of a biological particle onthe array of detection elements is substantially 1 or less, preferably ½or less, more preferably ¼ or less, more preferably 1/10 or less, morepreferably 1/50 or less, more preferably 1/100 or less, more preferably1/200 or less, relative to the ratio of the corresponding dimensions ofthe biological particle. In such situation the ratio of the shorter tothe longer of the two dimensions of the image of a biological particleon the array of detection elements is in certain circumstancessubstantially not the same within the area spanned by the array ofdetection elements.

Focusing—Collection Angle

The collection angle of a focusing arrangement used can have effect onthe intensity of any signal collected on the array of detectionelements. When high sensitivity is needed it is therefore practical toincrease the collection angle. The preferred size of the collectionangle can also be determined by other requirements which are made to thesystem, such as focusing depth. In these situations the collection angleof the focusing means is 15 degrees or less, preferably more than 15degrees, more preferably more then 30 degrees, more preferably more than60 degrees, more preferably more than 90 degrees, more preferably morethan 120 degrees, more preferably more than 150 degrees.

Detection Element—Size

The size of the detection elements determines to some extend itssensitivity. In some applications it is therefore of interest to havedetection elements of size of about 1 μm² or less. In certain situationsthe size of the detection elements in the array of detection elements isless than 20 μm², preferably less than 10 μm², more preferably less than5 μm², more preferably less than 2 μm², more preferably less than orequal to 1 m². In other situations the size of the detection elements inthe array of detection elements is greater than or equal to 5000 μm²,preferably greater than or equal to 2000 μm², more preferably greaterthan or equal to 1000 μm², more preferably greater than or equal to 500μm², more preferably greater than or equal to 200 μm², more preferablygreater than or equal to 100 and less than 200 μm², more preferablygreater than or equal to 50 and less than 100 μm², more preferablygreater than or equal to 20 and less than 50 μm².

Detection Element—Aspect Ratio

The aspect ratio of the detection elements can be important in thecollection of signals for the assessment of particles. A ratio of about1/1 is some times preferred, but under some conditions it can bepreferably to use ratio different from 1/1. In particular when thisfacilitates detection of signals from increased volume of any sample,thus allowing simultaneous assessment of more particles. In thosecircumstances the ratio of the shorter of the height or the width, tothe longer of the height or the width of the detection elements in thearray of detection elements is substantially equal or less than 1,preferably less than ½, more preferably less than ¼, more preferablyless than 1/10, more preferably less than 1/50, more preferably lessthan 1/100, more preferably less than 1/200.

Storage Capacity

Storage capacity, for instance used for storing information aboutmeasured signals from the detection elements, is often one of thosecomponents which have considerable effect on the cost of production. Itis therefore of interest to be able to perform the assessment ofparticles without substantial any use of such storage capacity, suchthat the assessment of biological particles in a sample is performedwithout the use of substantially any storage capacity means being usedto store measured signals from the detection elements in the array ofdetection elements.

On the other hand, it is often difficult to accomplish assessmentwithout the use of any storage capacity, but preferably the amount ofsuch storage capacity should not be more than what is needed to storethe information from all measured detection elements, preferably whereonly a fraction of the information can be stored.

In some situations measured signal from the detection elements in thearray of detection elements is stored by means of storage capacity, thestorage capacity being able to store a number of measurements equivalentto, or less than, the number of detection elements, preferably less than½ the number of detection elements, more preferably less than ¼ thenumber of detection elements, more preferably less than ⅛ the number ofdetection elements, more preferably less than 1/16 the number ofdetection elements, more preferably less than 1/32 the number ofdetection elements, more preferably less than 1/64 the number ofdetection elements, more preferably less than 1/128 the number ofdetection elements, more preferably less than 1/256 the number ofdetection elements, more preferably less than 1/512 the number ofdetection elements, more preferably less than 1/1024 the number ofdetection elements in the array of detection elements.

In other certain cicumstances it is advantageous that the measuredsignal from the detection elements in the array of detection elements isstored by means of storage capacity, the storage capacity being able tostore a number of measurements greater than the number of detectionelements, preferably equivalent to, or greater than, 2 times the numberof detection elements, more preferably equivalent to, or greater than, 4times the number of detection elements, more preferably equivalent to,or greater than, 8 times the number of detection elements, morepreferably equivalent to, or greater than, 16 times the number ofdetection elements, more preferably equivalent to, or greater than, 32times the number of detection elements, more preferably equivalent to,or greater than, 64 times the number of detection elements, morepreferably equivalent to, or greater than, 128 times the number ofdetection elements, more preferably equivalent to, or greater than, 256times the number of detection elements, more preferably equivalent to,or greater than, 512 times the number of detection elements, morepreferably equivalent to, or greater than, 1024 times the number ofdetection elements in the array of detection elements.

Other, more complicated aspects of the assessment of particles, canrequire the use of considerable amount of storage capacity. In thisaspect it can therefore be necessary to have storage capacity which canstore more information than is collected in one measurement of thedetection elements used.

Cuvette

A sample compartment, containing the sample being analysed, arrangespreferably as much sample volume as possible in such a way that it canbe exposed to the array of detection elements, thus allowing theanalysis of many particles simultaneously. One method for accomplishingthis, is to define the thickness of sample compartment in a directionwhich is not parallel to the plane of detection elements, thusincreasing the effective volume per are of sample compartment exposed tothe detection elements. The optimum thickness often being determined byany effective focus depth of a focusing system.

In such cases the sample compartment limits the dimension of the samplein the direction which is substantially not parallel to the plane ofarray of detection elements, to a thickness of 20 μm or less, preferablyto a thickness of more than 20 μm, more preferably to a thickness ofmore than 40 μm, more preferably to a thickness of more than 60 μm, morepreferably to a thickness of more than 80 μm, more preferably to athickness of more than 100 μm, more preferably to a thickness of morethan 140 μm, more preferably to a thickness of more than 180 μm, morepreferably to a thickness of more than 250 μm, more preferably to athickness of more than 500 μm, more preferably to a thickness of morethan 1000 μm.

Similarly, it is advantageous to extend the window of the samplecompartment in a direction parallel to the array of detection elements,thus increasing the effective area of the sample being exposed to thearray of detection elements. For some of these appications, the lengthof the dimension being 1 mm or more, preferably 2 mm or more, morepreferably 4 mm or more, more preferably 10 mm or more, more preferably20 mm or more, more preferably 40 mm or more, more preferably 100 mm ormore, more preferably 200 mm or more, more preferably 400 mm or more.

For some applications a tubular sample compartment is used whereby italso is possible to increase the number of particles being analysedsimultaneously by increasing the radius of such tubular samplecompartment. The optimum radius of such sample compartment is oftendetermined by the arrangement of the various components of the system,such as focus depth. The tube can in these circumstances have an innerradius of more than 0.01 mm, preferably 0.02 mm or more, more preferably0.04 mm or more, more preferably 0.1 mm or more, more preferably 0.2 mmor more, more preferably 0.4 mm or more, more preferably 1 mm or more,more preferably 2 mm or more, more preferably 4 mm or more, morepreferably 10 mm or more.

As mentioned above, the focus depth of the system, is often importantfor the determination of optimal dimensions of a sample compartment.Surprisingly it was found that it was possible to use dimension whichexceeded the focus depth of a focusing system, even to an extend wherethe dimension was greater than 1 times and less than 1.5 times thefocusing depth, more preferably equal to, or greater than 1.5 times andless than 2 times said focusing depth, more preferably equal to, orgreater than 2 times and less than 3 times said focusing depth, morepreferably equal to, or greater than 3 times and less than 4 times saidfocusing depth, more preferably equal to, or greater than 4 times andless than 6 times said focusing depth, more preferably equal to, orgreater than 6 times said focusing depth.

In the present specification and claims, the term “focus depth”designates the distance an object can move along the axis of a focusingsystem, without its image is distorted, such distortion being defined aswhen an image, which when in focus illuminates a single detectionelement, illuminates an area extending to 2 detection elements in one ortwo directions, when distorted. When two or more detection elements arecombined prior to analysis, the combined detection elements should beconsidered in the definition of focus depth.

The aspect ratio of a window region of the sample compartment can varyfrom about 1/1, preferably less than ½, more preferably less than ¼,more preferably less than 1/10, more preferably less than 1/20, morepreferably less than 1/33, more preferably less than 150, morepreferably less than 1/100, more preferably less than 1/200, morepreferably less than 1/500, more preferably less than 1/1000, morepreferably less than 1/2000, more preferably less than 1/4000, morepreferably less than 1/10000, depending on a focusing method, or otheraspects of other components of the system

The area of the exposing window can be as little as 0.01 mm² or more,preferably with an area of 0.1 mm² or more, more preferably with an areaof 1 mm² or more, preferably with an area of 2 mm² or more, preferablywith an area of 4 mm² or more, preferably with an area of 2 mm² or more,preferably with an area of 20 mm² or more, preferably with an area of 40mm² or more, more preferably with an area of 100 mm² or more, preferablywith an area of 200 mm² or more, preferably with an area of 400 mm² ormore, preferably with an area of 1000 mm² or more, preferably with anarea of 2000 mm² or more, preferably with an area of 4000 mm² or more,preferably with an area of 10000 mm² or more. The optimal are of thewindow often being defined by one or more aspects of this invention

Generally the volume of the sample being analysed should be as large aspossible. This allows the simultaneous assessment of a higher number ofparticles, but the optimal volume is often defined by one or moreaspects of this invention. For some applications according to theinvention the sample compartment limits the boundary of the sample inthree directions, in such a way that the volume of the sample is 0.01 μlor more, preferably 0.02 μl or more, more preferably 0.04 μl or more,more preferably 0.1 μl or more, more preferably 0.2 μl or more, morepreferably 0.4 μl or more, more preferably 1 μl or more, more preferably2 μl or more, more preferably 4 μl or more, more preferably 10 μl ormore, more preferably 20 μl or more, more preferably 40 μl or more, morepreferably 100 μl or more, more preferably 200 μl or more, morepreferably 400 μl or more.

In order to increase the effective volume of a sample being measured itcan be possible to include means in the sample compartment which canretain completely or partly the particles which are present in thesample. In this way it is possible to analyse a volume which issubstantially greater than the physical volume of the sample compartmentby sending the sample through the sample compartment prior to analysisand retaining particles from the sample volume inside the samplecompartment. Such means for retaining particles could be chemicallyactive means, electronical or magnetic field, or filter. In thesecircumstances it is preferred that at least one of the boundaries whichlimit the sample compartment, or a substantially flat surface containedsubstantially within the boundaries of the sample compartment is a meanswhich can retain particles being assessed, preferably a chemicallybinding means capable of binding particles, more preferably electronicor magnetic field means capable of withholding particles, morepreferably a filtering means capable of passing liquid sample or samplematerial and retaining particles.

In many preferred embodiments of the present invention, at least onedimension of the sample compartment could be so small that it could bedifficult for the sample to flow into or through the sample compartment.By using one aspect of the present invention it is possible to vary atleast one of the dimensions defining the sample compartment in such away that the dimension is substantially greater during the flowing ofthe sample into or through the sample compartment than during themeasurement of any signal from the sample. One effect of such avariation of at least one dimension of the sample compartment can be topartly or substantially completely replace the sample in the samplecompartment between the measurement of any signal from the sample. Suchembodiments could be where at least one of the dimensions definingboundaries which limit the sample compartment before or during theintroduction of the sample to the sample compartment, is substantiallydifferent from the dimension during the measurement of any signal fromthe sample, preferably where the dimension is substantially greaterbefore or during the introduction of the sample to the samplecompartment than during the measurement of any signal from the sample,preferably where the dimension being varied is substantially notparallel to the plane of array of detection elements, preferably wherethe effect of the difference in the dimension is to replace at least apart of the sample in the sample compartment with a different partbetween measurement of any signal from the sample, preferably where theeffect of the difference in the dimension is to improve the flowing ofthe sample into or through the sample compartment.

Sample Pre-Treatment

Often it is preferred to analyse a sample of a sample material withoutsubstantially any modification of the sample in full or in part. Otherconditions are favoured by imposing one or more modification upon thesample prior to measurement, for instance by removing interferingcomponents or phenomena, or by allowing some modification of a particleor a part of a particle prior to measurement.

In other circumstances the sample, or parts of said sample, beinganalysed has been given a chemical, a mechanical or a physical treatmentprior to analysis. This treatment could be one or several of thefollowing: exposure to gravity and/or centrifugation, filtering,heating, cooling, mixing, sedimentation, salvation, dilution,homogenisation, sonification, crystallisation, chromatography, ionexchange, electrical field, magnetic field, electromagnetic radiation.The effects of the treatment will normally be an enhancement of anysignal observed from the sample used in the assessment of biologicalparticles in the sample, and/or suppression of any interfering signal.

In some of the embodiments of the invention the temperature of thesample can be controlled, either by addition or removal of heat from thesample and the temperature of the sample during measurement of thebiological particle containing sample is between 0° C. and 90° C., morepreferable between 5° C. and 90° C., more preferable between 10° C. and90° C., more preferable between 20° C. and 90° C., more preferablebetween 25° C. and 90° C., more preferable between 30° C. and 90° C.,more preferable between 35° C. and 90° C., more preferable between 40°C. and 90° C.

In one situation of assement the temperature of the sample is controlledby the ambient temperature and the temperature of the sample duringmeasurement of the biological particle containing sample is between 0 Cand 90° C., more preferable between 5° C. and 90° C., more preferablebetween 10° C. and 90° C., more preferable between 20° C. and 90° C.,more preferable between 25° C. and 90° C., more preferable between 30°C. and 90° C., more preferable between 35° C. and 90° C., morepreferable between 40° C. and 90° C.

Colouring of Objects

Often the particles in question exhibits properties which facilitate thedetection of a signal which can be used for the assessment, butsometimes it is preferred to add one or more type of molecules in orderto enhance or facilitate any detection of a signal. The number ofdifferent types of molecules added depends on the complexity of theassessment, and on the nature of the particles and sample material beinganalysed. It is for instance often advantageous to use two or more suchas 3 or even 4 types of molecules when the assessment concerns theidentification of two or more types of particles, where the differentparticles interact differently with the different molecules, forinstance by giving rise to a fluorescent signal at different wavelength.Often the addition of such two or more types of molecules is donesimultaneously, but under some conditions it is preferred to add themolecules at different times, preferably in such a way that one or moremeasurements are carried out between the addition of molecules. Theseadded molecules can interact with the particles for instance by beingretained within them, interacting with them or being prepelled by them.The molecules being intentionally added to the sample before or duringthe measurement, preferably one at a time, more preferably more than oneat a time.

It is preferred that at least one of the types of molecules is added toa first sample of a sample material and at least another of the types ofmolecules is added to a second sample of the sample material, preferablywhere the number of samples of a sample material is equal or less to thenumber of said different types of molecules, and where at least onemeasurement is taken from each sample.

In situations where an initial measurement is preferred, the initialmeasurement is one taken with any intentionally added molecules, ameasurement of a sample is taken before at least one of said moleculeshave been added to the sample and at least one measurement of the sampleis taken after all said types of molecules have been added.

The intentionally added molecules can give rise to one or several of thefollowing phenomena assisting in the assessment of biological particles:attenuation of electromagnetic radiation, photoluminescence whenilluminated with electromagnetic radiation, scatter of electromagneticradiation, raman scatter.

The assessment of biological particles can be based on the use ofnucleic acid dye as an intentionally added molecule in an amount of morethan 30 μg per ml of the sample, more preferable in an amount of lessthan 30 μg per ml of the sample, more preferable in an amount of lessthan 20 μg per ml of the sample, more preferable in an amount of lessthan 10 μg per ml of the sample, more preferable in an amount of lessthan 5 μg per ml of the sample, more preferable in an amount of lessthan 2 μg per ml of the sample, more preferable in an amount of lessthan 1 μg per ml of the sample, more preferable in an amount of lessthan 0.3 μg per ml of the sample, more preferable in an amount of lessthan 0.03 μg per ml of the sample, more preferable in an amount of lessthan 0.003 μg per ml of the sample, more preferable in an amount of lessthan 0.0003 μg per ml of the sample, the nucleic acid stain being one orseveral of the following, but not limited to: phenanthridines (e.g.ethidium bromide CAS-1239-45-8, propidium iodide CAS-25535-16-4),acridine dyes (e.g. acridine orange CAS-65-61-2/CAS-10127-02-3), cyaninedyes (e.g. TOTO™-1 iodide CAS#: 143 413-84-7-Molecular Probes, YO-PRO™-1iodide CAS#: 152 068-09-2-Molecular Probes), indoles and imidazoles(e.g. Hoechst 33258 CAS#: 023 491-45-4, Hoechst 33342 CAS#: 023491-52-3, DAPI CAS#:28718-90-3, DIPI(4′,6-(diimidazolin-2-yl)-2-phenylindole)).

The assessment of biological particles can be based on the use ofpotentiometric membrane dye as an intentionally added molecule in anamount of either more than 30 μg per ml of the sample, or, morepreferable in an amount of at the most 30 μg per ml of the sample, morepreferable in an amount of less than 20 μg per ml of the sample, morepreferable in an amount of less than 10 μg per ml of the sample, morepreferable in an amount of less than 5 μg per ml of the sample, morepreferable in an amount of less than 2 μg per ml of the sample, morepreferable in an amount of less than 1 μg per ml of the sample, morepreferable in an amount of less than 0.3 μg per ml of the sample, morepreferable in an amount of less than 0.03 μg per ml of the sample, morepreferable in an amount of less than 0.003 μg per ml of the sample, morepreferable in an amount of less than 0.0003 μg per ml of the sample, thenucleic acid stain being one or several of the following, but notlimited to: Rhodamine-123, Oxonol V.

In order to assure fast assessment of a sample it is of interest to beable to perform analysis shortly after the mixing of any chemicalcomponents with sample. This time should therefore be less than 60seconds, or preferably less than 30 seconds or even as low as 15 secondsand in other preferred situations as low as 10 seconds, and preferablyas short as 2 seconds or less and even shorter than 1 second.

One useful method for the introduction of chemical components to thesample is to place one or more chemical component in a container. Thecontainer should then be connected to a flow system where the sampleflows, and at least a portion of the sample flown through the chemicalcontainer and thus allowing the mixing of the chemical components withthe sample. In order to control the use of chemical components it is ofinterest to limit the amount of chemical components to substantially theamount needed for the analysis. The chemical components could be on theform of a liquid solution or suspension, as liquid, or solid. Ofparticular interest would be to have the chemical components on a formwhich would allow fast mixing with the sample, for instance by usingfreeze dried matter. The possibility of being able to replace thechemical container with another chemical container between analysis isof interest in order to assure reproducible addition of chemicalcomponents in the measurement of each sample.

Variation in Addition

When performing a quantitative assessment of particles it is normallynecessary to control the addition of any component to the sample, inorder not to affect the result of the assessment. The present inventionoffers embodiments where such requirements are less important than underconventional situations. This can be accomplished by introducing thecomponents on a form which has only limited effect on the assessment,such as introducing any component as solid matter, thereby substantiallynot altering the volume of any sample being analysed, even though thefinal concentration of any added component displays considerablevariation. Further it is possible that the variation in theconcentration of one or more intentionally added component(s) or one ormore intentionally added molecules in a sample, is less than, or equalto 1%, preferably more than 1%, more preferably more than 2%, morepreferably more than 5%, more preferably more than 10%, more preferablymore than 25%, more preferably more than 50%, of the averageconcentration of said component when expressed as 1 standard deviation.

Flow Conditions

In a preferred embodiment of the invention the particles being assessedare substantially at stand-still during measurement, thus allowing theoptimal use of measurement time in order to improve any signal to noiseconditions. This arrangement also eliminates any error which could beinherent in the assessment of particles caused by variation in flowconditions, particularly when an assessment of a property is a volumerelated property such as the counting of particles in a volume ofsample.

In other preferred embodiments the particles are moving duringmeasurement, thus producing the image of a moving particle on the arrayof detection elements. This can offer advantage in the assessment ofparticles, especially when any image of the movement can be used for theidentification of a particle. Such movement of image can be homogeneousthroughout the array of detection elements, or it can be varying forinstance depending on the position of the particle within the samplecompartment.

It is also possible to have movements of image, consisting of more thanone directional component, which can give advantage when it is necessaryto distinguish a particle travelling in a predefined way, from abackground signal which is substantially random.

When applying a relative movement between the sample and the array ofdetecting elements, either by physically moving the sample or by movingthe image of the sample relative to the array by, e.g., optical orcomputer means, the rate of the movement will normally be adapted to theeffect to be obtained. Thus, e.g., where the concentration of a type ofparticle to be counted is very low, it may be advantageous to pass alarge volume of sample through af flow system during one exposure, inorder to increase the chance that one or more particles will in fact bedetected by the array.

The movement of the sample can preferably be accomplished by applying apositive or negative pressure difference across the sample compartment.Such pressure difference can be created by one or several means such asperistaltic pump, piston pump, membrane pump, or syringe.

Flow System

When a sample compartment is substantially mechanically fixed in ameasuring system, it is an advantage to make use of a method of flowsystem, which is capable to flow the sample, and/or any other liquid orcomponent into the sample compartment through an inlet, and out of thesample compartment through an outlet, possibly using the inlet foroutlet and thereby reducing the complexity of any flow system. Any suchflow is often controlled by the use of one or more valves which cancontrol the flow of sample or any other component. Preferably where theflow of liquid in the sample compartment is brought about by a pump,said pump being situated either upstream to the sample compartment ordownstream to the sample compartment, the pump being one or several ofthe following: peristaltic pump, piston pump, membrane pump, centrifugalpump, hypodermic syringe. Other types of pump could of course be usedfor this specific topic, but the ones listed above are the ones normallyused.

In other preferred situations the flow of liquid in the samplecompartment can brought about by a vacuum, the vacuum being applied froma reservoir having a low pressure before the analysis. The vacuum can beestablished by a mechanical or physical action creating the vacuumsubstantially simultaneously with the introduction of the sample. Thesemechanical or physical actions can be: a peristaltic pump, a pistonpump, a membrane pump, a centrifugal pump and a hypodermic syringe.

Due to the fact that flow only in one direction is preferred, it is ofparticular interest to use valves which substantially only allow theflow in one direction. Such valves can be placed up- and/or downstreamfrom the sample compartment. One effect of the use of such valves couldbe to confine at least a part of the sample in a flow system.

When other components are added to the sample this can be accomplishedby means of a flow system which can mix two or more streams of liquid.

In a preferred embodiment of the invention this flow system allows themixing of the sample material with a solid material which preferably isa mixture of two or more chemical component. The solid material ispreferably a freeze dried material.

After any measurement has been carried out, it is preferred that anysample, or other component used being directed to a waste reservoirwhich is substantially closed to prevent spilling or evaporation fromthe reservoir whereby a substantially closed flow system is providedaccording to the invention.

The outlet from the sample compartment is passed through a flowcontrolling means, such as a valve, which only allows fluid in gas phaseto pass through. One such type of valves which often is preferred, isone which allows gas and air to pass but can close when the valve comesin contact with liquid sample.

Disposable Cuvette

Another aspect of the present invention, which is particularly ofinterest when the sample, or any component added to the sample can beconsidered hazardous, or difficult to handle, is the use of a removablesample compartment. Such sample compartment is readily removed from themeasuring system, allowing another sample compartment to take its place.Preferably such sample compartments can be reused or regenerated, maybeafter rinsing.

One interesting aspect of a replaceable sample compartment is thepossibility of a method for the substantial irreversible closing of thesample compartment after the addition of a sample or any othercomponents, thus preventing any accidental spill or leakage from thesample compartment during storing or transport.

Such a replaceable sample compartment is preferably with substantiallyno connection to the flow system during analysis and the samplecompartment can preferably be removed from the sensing area betweenobservations for the purpose of replacing the sample within the samplecompartment and/or preferably for the purpose of replacing the samplecompartment with another sample compartment which preferably containinganother sample.

The sample compartment can in some situations be used for the analysisof limited number of samples or sample materials, preferably less than10, more preferably less than 5, more preferably less than 2, morepreferably only 1, before said removable sample compartment beingsubjected to emptying and/or rinsing and/or addition of one or morecomponents.

In situations where no spilling is preferred and/or where the removablesample compartment is intended to be used only for measurement of onesample or sample material it is preferred that any acces to theremovable sample compartement is substantially irreversibly closed priorto, during or after analysis, preferably in such a way that any part ofthe sample material, or any component added to the sample material cannot be removed from the removable sample compartment after it has beenintroduced therein.

When the compartment is intended to be destroyed or re-used after use itis preferred that the compartment is made up by a material which allowsdestruction by means such as burning or illumination by electromagneticradiation. In the situations where the destruction comprisses a re-useof the material from which the compartment is made of, a process ofregeneration of the materials is preferred to comprisse one or severalof the following steps: emptying the sample compartment for any samplematerial or any other components, rinsing or washing, removal of one ormore physical component of the sample compartment, replacing of one ormore physical component of the sample compartment or addition of one ormore chemical component.

When addition of a chemical to a sample in the sample compartment it ispreferred that the sample compartment can comprisse one or morecompartments where chemical or physical components can be stored suchthat the chemical or physical components can be added to any samplepresent in the sample compartment one at a time or more than one at atime. In this way, the sample compartment can be formed in such a waythat it comprises more than one compartments where a portion of the samesample material, or portions of different sample material, or portion ofother components can be placed. This is also of interrest where, forinstance, the assement involve or allow a controlled mixing of liquids.

A sample compartment with more than one compartment could also allow theanalysis of more than one portion of the same sample material, or theanalysis of more than one sample materials by allowing the differentcompartment to be exposed to the array of detection elements.

One aspect of such removable sample compartment is that more than oneportions of the same sample material can be subjected to analysis byexposure to the array of detection elements. This can be done byallowing the sample compartment to be moved, thus exposing a differentportion of the sample compartment, or by allowing the sample within thesample compartment to flow and thereby substantially replace any samplevolume exposed with a different sample volume.

This invention offers also methods for the assessment of particles in aremovable sample compartment, where more than one such samplecompartments are loaded with sample and placed in a transport meanswhich can move the different sample compartment in a position whichallows exposure of signals to the array of detection elements. Thisallows substantial automation of the assessment of particles since morethan one sample can be handled at once.

One preferred implementation of the method is one which allows thesubstantially simultaneous assessment of more than one sample. This canbe accomplished by placing two or more, or even four or more preferablyindependent measuring systems, comprising at least one samplecompartment each, in one disposable sample unit. The signals from thetwo or more sample compartments can be measured one at a time, or two ormore simultaneously.

Sample Volume

In the present section and other sections of this part of thedescription, the term “sample” does not necessarily mean the samplepresent in the compartment, but rather the sample introduced into a flowsystem used according to the invention. It is of interest to minimisethe use of sample material and any chemical component used for theanalysis. This can be accomplished by the use of the present invention.Sample volumes as small as 5 ml or less and even as small as 0.02 ml canbe used. The volume of the sample needed is highly dependent on thenumber of particles present in the sample and the predeterminedstatistical quality parameter sought, whereby typical volumes applied isless than 5 ml of a liquid sample, preferably by using less than 2 ml ofa liquid sample, more preferably by using less than 1 ml of a liquidsample, more preferably by using less than 0.5 ml of a liquid sample,more preferably by using less than 0.2 ml of a liquid sample, morepreferably by using less than 0.1 ml of a liquid sample, more preferablyby using less than 0.05 ml of a liquid sample, more preferably by usingless than 0.02 ml of a liquid sample, more preferably by using less than0.01 ml of a liquid sample, the volume being defined as the total volumeof any liquid sample introduced to the sample compartment, or any flowsystem connected to the sample compartment before or after or during themeasurement of the sample.

Preferred embodiments of the present invention make it possible toassess particles from a considerably large volumes of sample. This canallow the measurement of samples with only few particles of interest pervolume of sample. Sample volumes larger than 10 ml and even larger than100 ml can be used for the analysis. using more than 1 ml of a liquidsample, preferably by using more than 2 ml of a liquid sample, morepreferably by using more than 3 ml of a liquid sample, more preferablyby using more than 5 ml of a liquid sample, more preferably by usingmore than 10 ml of a liquid sample, more preferably by using more than20 ml of a liquid sample, more preferably by using more than 50 ml of aliquid sample, more preferably by using more than 100 ml of a liquidsample, the volume being defined as the total volume of any liquidsample introduced to any flow system connected to the sample compartmentbefore or after or during the measurement of the sample.

Sampling

Large volume of the sample can be measured by passing the volume ofsample through a particle retaining means, such as filter, electricalfield, magnetic field, gravitational field. When the particles from alarge sample are retained, those particles can be resuspend in a volumewhich is less than the volume of sample passed through the particleretaining means, preferably where the volume used for the resuspensionis only ½

the volume passed through the particle retaining means, more preferably¼ or less the volume passed through the particle retaining means, morepreferably ⅛ or less the volume passed through the particle retainingmeans, more preferably 1/20 or less the volume passed through theparticle retaining means, more preferably 1/50 or less the volume passedthrough the particle retaining means, more preferably 1/100

or less, or even only 1/100 or less the volume which originally waspassed through the particle retaining means. The particle retainingmeans should preferably be able to retain substantially all particlespresent in a sample, or at least a substantially representative fractionof at least one type of particles present in the sample.

In one embodiment of the present invention a signal from the particlesbeing analysed is detected while the particles are still substantiallyretained by a particle retaining means. In such embodiment the particleretaining means are integrated with, or in close connection to a samplecompartment.

In the following, the information is given as numbered items, startingwith the arbitrary item number 87:

Multiple Exposure

#87. A method according to any of the preceding items, where theassessment of the biological objects is based on observation from 2,preferably more than 2 and less than 4, more preferably more than orequal to 4 and less than 8, more preferably more than or equal to 8 andless than 16, more preferably more than or equal to 16 and less than 32,more preferably more than or equal to 32 and less than 64, morepreferably more than or equal to 64 and less than 128, more preferablymore than or equal to 128 and less than 256, more preferably more thanor equal to 256 and less than 512, more preferably more than or equal to512 and less than 1024, more preferably more than or equal to 1024measurement periods.

#88. A method according to item 87, where at least one of themeasurement periods is divided up into at least two periods, where in atleast one of the periods the array of detection element is substantiallyexposed with signals from the sample and where in at least one of theperiods the array of detection elements are substantially not exposed tosignals from the sample, the periods being controlled by means which canactivate the transmission of electromagnetic radiation on the sample, orthe emission of, or the transmission of electromagnetic radiation fromthe sample.

#89. A method according to items 87 or 88, where the number of theactive periods within a measurement period are 2, preferably 3, morepreferably 4, more preferably more than 4 and less than 8, morepreferably 8 or more and less than 16, more preferably 16 or more andless than 32, more preferably 32 or more and less than 64, morepreferably 64 or more.

#90. A method according to any of the items 87 through 89, where eachdetection element in the array of detection elements measures signalfrom substantially the same fraction of the sample in two or more of theactive periods within a measurement period.

#91. A method according to any of the items 87 through 90, where eachdetection element in the array of detection elements measures signalfrom substantially different fraction of the sample, preferably where nofraction of the sample is measured by more than one detection elementson the array of detection elements, in two or more of the active periodswithin a measurement period.

#92. A method according to any of the items 87 through 91, whereduration of the measurement periods is shorter than or equal to 1×10⁻⁶seconds, preferably longer than 1×10⁻⁶ seconds and shorter than 1×10⁻⁵seconds, more preferably longer than 1×10⁻⁵ seconds and shorter than1×10⁻⁴ seconds, more preferably longer than 1×10⁻⁴ seconds and shorterthan 1×10⁻³ seconds, more preferably longer than 1×10⁻³ seconds andshorter than 1×10⁻² seconds, more preferably longer than 1×10⁻² secondsand shorter than 1×10⁻¹ seconds, more preferably longer than 1×10⁻¹seconds and shorter than 1 second, more preferably longer than 1 secondand shorter than 10 seconds, more preferably longer than 10 seconds.

#93. A method according to any of the items 87 through 92, whereduration of all the measurement periods is substantially equal.

#94. A method according to any of the items 87 through 92, whereduration of at least 2 said measurement periods is substantiallydifferent.

#95. A method according to any of the items 87 through 94, where eachdetection element in the array of detection elements measures signalfrom substantially the same fraction of the sample in two or more of themeasurement periods.

#96. A method according to any of the items 87 through 95, where eachdetection element in the array of detection elements measures signalfrom substantially different fraction of the sample, preferably where nofraction of the sample is measured by more than one detection elementsin the array of detection elements, in two or more of the measurementperiods.

Detection Error

#97. A method according to any of the preceding items, which can assessthe number of biological particles in a sample with total error,expressed as standard prediction error which is more than or equal to30%, preferably less than 30%, more preferably less than 20%, morepreferably less than 10%, more preferably less than 6%, more preferablyless than 4, more preferably less than 2%, more preferably less than 1%of the average value of number of biological particles per volumesample.

Sample Throughput

#98. A method according to any of the preceding items, where theassessment of biological particles can be carried out at a rate which isless than or equal to 10 assessments per hour, preferably greater than10 assessments per hour, more preferably greater than 30 assessments perhour, more preferably greater than 50 assessments per hour, morepreferably greater than 100 assessments per hour, more preferablygreater than 200 assessments per hour, more preferably greater than 300assessments per hour, more preferably greater than 400 assessments perhour, more preferably greater than 500 assessments per hour, morepreferably greater than 600 assessments per hour, more preferablygreater than 700 assessments per hour, more preferably greater than 1000assessments per hour.

#99. A method according to any of the preceding items, which uses 2,preferably 3, more preferably 4, more preferably more than 4 paralleldetection systems for the substantially simultaneous assessment ofbiological particles in a sample.

Detection Limits

#100. A method according to any of the preceding items, where theassessment of biological particles in a sample is carried out when thenumber of biological particles in the sample material is grater than1×10⁸, preferably less than or equal to 1×10⁸, more preferably less than1×10⁷, more preferably less than 1×10⁶, more preferably less than 1×10⁵,more preferably less than 1×10⁴, more preferably less than 1×10³, morepreferably less than 1×10², more preferably less than 10, morepreferably less than 1, more preferably less than 0.1, per ml sample.

Signal Source

#101. A method according to any of the preceding items, where the signalwhich is detected is substantially caused by one or several of thefollowing: photoluminescence with lifetime of the exited state of lessthan or equal to 10⁻⁶ seconds, photoluminescence with lifetime of theexited state of garter than 10⁻⁶ seconds, chemiluminescence, rayleighscatter, raman scatter, attenuation of electromagnetic radiation,absorption of the electromagnetic radiation, scatter of theelectromagnetic radiation.

Wavelength Sensitivity

#102. A method according to any of the preceding items, where the arrayof detection elements is sensitive to electromagnetic radiation ofwavelength in one or several of the following regions: 100 m to 200 nm,200 nm to 600 nm, 300 nm to 700 nm, 400 nm to 800 nm, 600 nm to 1 μm,800 nm to 2 μm, 2 μm to 10 μm, 5 μm to 10 μm, 10 μm to 20 μm, 20 μm to40 μm.

Wavelength Separation

#103. A method according to any of the preceding items, where spectrallyrich electromagnetic radiation can be separated into substantially 1wavelength component or waveband which is transmitted onto the sample,preferably into 2 or more wavelength components or wavebands which aretransmitted onto the sample, one at a time or two or moresimultaneously.

#104. A method according to any of the preceding items, spectrally richelectromagnetic radiation emitted from, or transmitted through thesample is separated into substantially 1 wavelength component orwaveband, which is measured by a detection element in said array ofdetection elements, preferably into 2 or more wavelength components orwavebands, which are measured by a detection element in the array ofdetection elements, one at a time or two or more simultaneously.

#105. A method according to any of the preceding items, where spectrallyrich electromagnetic radiation transmitted onto the sample is spatiallyseparated into a plurality of wavelength components, in such a way thatat least two fractions of the sample, are exposed to substantiallydifferent wavelength components.

#106. A method according to any of the preceding items, where spectrallyrich electromagnetic radiation emitted from, or transmitted through thesample is spatially separated into a plurality of wavelength components,in such a way that each of the detection elements in the array ofdetection elements, measuring information from substantially the samefraction of the sample, is exposed to substantially different wavelengthcomponents.

#107. A method according to any of the items 103 through 106, where theseparation of spectrally rich electromagnetic radiation is brought aboutby one or several of the following, but not limited to: interferencefilters, coloured filters, an optical grating, a prism, an opticallyactive crystals.

#108. A method according to any of the preceding items, whereelectromagnetic radiation which is transmitted onto, or emitted from, ortransmitted through the sample is intensity modulated.

#109. A method according to any of the preceding items, whereelectromagnetic radiation which is transmitted onto, or emitted from, ortransmitted through the sample is modulated by optically active crystalsor interferometry, preferably by the use of a Michelson interferometer,more preferably by the use of an interferometer where at least onereflecting surface can be moved.

Light Sources

#110. A method according to any of the preceding items, where thetransmission of electromagnetic radiation onto the sample isaccomplished by the use of illuminating means.

#111. A method according to item 110, where the illumination means are 2or more, preferably 3 or more, more preferably 4 or more, morepreferably 6 or more, more preferably 8 or more, more preferably 10 ormore, light emitting diodes preferably emitting electromagneticradiation of substantially the same wavelength band.

#112. A method according to item 110 or 111, where the electromagneticradiation transmitted onto the sample is focused by a focusing mean, thefocusing mean having the effect of substantially increasing theintensity of said electromagnetic radiation in or at said sample.

#113. A method according to any of the items 110 through 112, where theelectromagnetic radiation transmitted onto the sample is accomplished bytwo or more illuminating means, at least two of the illuminating meanshaving substantially different radiation properties in at least onewaveband, the illuminating means being operated in such a way that alltransmit substantially simultaneously, preferably at least one of theilluminating means transmitting while at least one other of theilluminating means is not transmitting, more preferably where only oneof the illuminating means is transmitting at a time.

#114. A method according to any of the items 110 through 113, where theilluminating means are one or several of the following, but not limitedto: light emitting diodes, lasers, laser diodes, thermal light source,gas discharge lamp.

Reflection

#115. A method according to any of the items 110 through 114, where atleast a portion of electromagnetic radiation which is transmittedthrough a sample is reflected back onto or through the sample by the useof a reflecting means, preferably including reflectance means which alsocan reflect electromagnetic radiation which is scattered or reflectedfrom the boundaries of the sample compartment or the sample is reflectedback onto the sample, more preferably where said reflectance means aresubstantially included in the means which define the boundaries of saidsample compartment, preferably where the reflectance mean is one orseveral dichroic mirrors.

Incidence Angle.

#116. A method according to any of the items 110 through 115, where theelectromagnetic radiation is transmitted onto said sample from aposition which forms an angle which is substantially 0 degrees,preferably between 0 and 15 degrees, more preferably between 14 and 30degrees, more preferably between 29 and 45 degrees, more preferablybetween 44 and 60 degrees, more preferably between 59 and 75 degrees,more preferably between 74 and 90 degrees, from the direction betweensaid sample and said array of detection elements.

#117. A method according to any of the items 110 through 116, where theelectromagnetic radiation is transmitted onto said sample from aposition which forms an angle which is substantially 90 degrees, fromthe direction between said sample and said array of detection elements.

#118. A method according to any of the items 110 through 117, where theelectromagnetic radiation is transmitted onto said sample from aposition which forms an angle which is between 106 and 90 degrees,preferably between 121 and 105 degrees, more preferably between 136 and120 degrees, more preferably between 151 and 135 degrees, morepreferably between 166 and 150 degrees, more preferably between 180 and165 degrees, more preferably substantially 180 degrees, from thedirection between said sample and said array of detection elements.

Detector Types

#119. A method according to any of the preceding items, where said arrayof detection elements is one or several of the following types: fullframe CCD, frame transfer CCD, interline transfer CCD, line scan CCD.

#120. A method according to any of items #1 through #118, where saidarray of detection elements is a CMOS image sensor, preferably a CMOSimage sensor with on-chip integrated signal condition and/or signalprocessing, more preferably a CMOS image sensor with on-chip integratedcomputing means capable of performing image processing.

Signal Conditioning—Software

#121. A method according to any of the preceding items, where a measuredsignal from one or more detection elements is corrected for systematicor varying bias by the use of a calculating means, the bias correctionbeing accomplished by the use of one or more pre-defined value(s),preferably where each measured signal for one or more detection elementsin said array of detection elements has one or more pre-definedvalue(s), more preferably where each pre-defined value is determined onthe bases of one or more of any previous measurements.

#122. A method according to item 121 where the bias correction isperformed by subtracting the results obtained in one or several of othermeasurements from the measured signal, preferably where the othermeasurements are one or several of measurements of the same sample, orsample material, more preferably where the other measurement is themeasurement taken previously of the same sample or sample material.

#123. A method according to any of the preceding items, where a measuredsignal from one or more detection elements is corrected for intensity bythe use of a calculating means, said correction being accomplished bythe use of one or more pre-defined value(s), preferably where eachmeasured signal for one or more detection elements in said array ofdetection elements has one or more pre-defined value(s), more preferablywhere each pre-defined value is determined on the bases of one or moreof any previous measurements.

#124. A method according to any of the preceding items, where theassessment of biological particles is done on the bases of twomeasurements of the same sample, or sample material, where the twomeasurements are combined by subtracting one of the measurements fromthe other measurements thereby creating a measurement result wheresignals occurring in only one of the measurements are represented byeither a positive or negative measurement result, and signals occurringin both measurements are represented by substantially zero measurementresult, preferably using only positive measurement results in theassessment of biological particles, more preferably using both positiveand negative measurement results in the assessment of biologicalparticles, more preferably using the absolute value of the measurementresults in the assessment of biological particles.

#125. A method according to item 124 where two measurement results arecombined by simple addition, preferably where three measurement resultsare combined, more preferably where four measurement results arecombined, more preferably where five measurement results are combined,more preferably where six measurement results are combined, morepreferably where more than six measurement results are combined, andused in the assessment of biological particles.

Assessment—One dimension

#126. A method according to any of the preceding items, where thedistinction between signals from particles and signal from samplebackground is based on substantially simultaneous use of more than 1,preferably 2 or more, more preferably 4 or more, more preferably 8 ormore, more preferably 16 or, more preferably 32 or, more preferably 64or more measured signals and/or bias corrected signals and/orsensitivity corrected signals from said detection elements in said arrayof detection elements.

Assessment—Two dimensions

#127. A method according to any of the preceding items, where signalsfrom more than 1, preferably more than 4, more preferably 10 or more,more preferably 50 or more, more preferably 100 or more, more preferably200 or more substantially parallel, substantially straight lines ofdetection elements are used for substantially simultaneous distinctionbetween signal from particles and signal from sample background,preferably by combining the signals from said substantially straightlines into one array of values, each value being obtained by combiningone or more signals from substantially each straight lines of detectionelements thus allowing data from two dimensional array of detectionelements to be analysed in the same manner as data from one dimensionalarray of detection elements.

#128. A method according to any of the preceding items, where the resultfrom the distinction between signal from particles and sample backgroundof the number of objects in 1, preferably 2 or more, more preferably 4or more, more preferably 8 or more line(s) is/are used in the assessmentof biological particles in an adjacent line of detection elements.

Qualitative Assessment

#129. A method according to any of the preceding items, where theassessment of biological particles in a sample is used to confirm thepresence of any predetermined biological particles in said sample.

Image Processing

#130. A method according to any of the preceding items, where theassessment of biological particles is done by subjecting signals fromtwo dimensional array of detection elements to the methods of imageprocessing or image analysis.

Power

#131. A method according to any of the preceding items, where the sourceof electrical power is a transformer, capable of transformingalternating electrical source with alternating voltage between −150 and150 volt, or with alternating voltage between −250 and 350 volt, or withalternating voltage between −350 and 350 volt, into substantially directcurrent voltage.

#132. A method according to any of the preceding items, where the sourceof electrical power is one of several of: an accumulator, a removableaccumulator, a battery, a rechargeable battery.

Objects

#133. A method according to any of the preceding items, where thebiological particles are somatic cells and the liquid sample material ismilk.

#134. A method according to any of the items 1 through 132, where thebiological particles are bacteria and the liquid sample material ismilk.

#135. A method according to any of the items 1 through 132, where thebiological particles are bacteria and the liquid sample material isblood.

#136. A method according to any of the items 1 through 132, where thebiological particles are somatic cells and the liquid sample material isblood.

#137. A method according to any of the items 1 through 132, where thebiological particles are bacteria and the liquid sample material isurine.

#138. A method according to any of the items 1 through 132, where thebiological particles are somatic cells and the liquid sample material isurine.

#139. A method according to any of the items 1 through 132, where thebiological particles are bacteria and the liquid sample material iswater.

#140. A method according to any of the items 1 through 132, where thebiological particles are blood cells and the liquid sample material isblood.

#141. A method according to any of the items 1 through 132, where thebiological particles are blood platelets and the liquid sample materialis blood.

Application

#142. A method according to any of the items 1 through 134, where theassessment is the determination of the number of somatic cells in avolume of milk or a milk product, the type of the milk or milk productbeing one or several of the following: cow milk, goats milk, sheep milk,or buffalo milk.

#143. A method according to any of the items 1 through 134, where theassessment is the determination of the number of bacteria in a volume ofmilk or a milk product, the type of the milk or milk product being oneor several of the following: cow milk, goats milk, sheep milk, orbuffalo milk.

#144. A method according to any of the items 1 through 134, where theassessment is the determination of the types of bacteria in a volume ofmilk or a milk product, the type of the milk or milk product being oneor several of the following: cow milk, goats milk, sheep milk, orbuffalo milk.

#145. A method according to any of the items 142 through 144, where saidassessment is carried out substantially simultaneously with the milking,preferably by including the system at-line, more preferably by includingthe system in-line with a milking system.

#146. A method according to any of the items 142 through 145, where themilk sample is collected during milking, preferably in such a way thatthe composition of the sample is substantially a representation of thecomposition of the entire milk being milked, the milk being collected ina container unit, preferably where the container unit also contains atleast one sample compartment, the milk sample or a portion of the milksample being flown into the sample compartment upon completion of themilking.

#147. A method according to any of the items 142 through 146, where theresults of the assessment are transferred to one or several informationstorage means, preferably the information storage means also being ableto store other information about the milking, more preferably theinformation storage means also being able to store information about thebulk of milk previously collected.

#148. A method according to any of the items 142 through 147, where theinformation storage means includes means to indicate whether the milkbeing milked should be directed to one or several of storage facilitiesor outlet, the indication being based on the assessment of the number ofsomatic cells per volume, preferably the indication being based on theassessment as well as other information present in the informationstorage means about milking of individual animals or the bulk of milk,the other information being one or several of, but limited to:conductivity, impedance, temperature, fat content, protein content,lactose content, urea content, citric acid content, ketone content,somatic cell count.

#149. A method according to any of the items 142 through 148, where thepurpose of the direction of any milk being milked to one or several ofstorage facilities or outlets is to adjust the properties of any bulk ofmilk, preferably with regard to the number of somatic cells per volume.

#150. A method according to any of the items 142 through 149, where theassessment is carried out after the milking has taken place, preferablythe milk being substantially not altered before measurement.

#151. A method according to any of the items 142 through 149, where theassessment is carried out after the milking has taken place, the milkbeing modified before measurement, preferably in such a way that themodification extends the durability of the sample material, themodification being one or several of, but not limited to; addition ofone or more chemical component which substantially inhibits bacterialgrowth in the sample material, addition of one or more chemicalcomponent which substantially inhibits the growth of fungus, addition ofone or more chemical component which has colouring properties saidcolouring being used to aid visual identification of the milk.

#152. A method according to any of the items 142 through 151, where theassessment is carried out substantially simultaneously with theassessment of the amount of any constituent in said sample material,preferably by using substantially a same portion of the sample materialfor the assessment, said constituent being one or several of, but notlimited to: fat, protein, lactose, urea, citric acid, glucose, ketones,carbon dioxide, oxygen, pH, potassium, calcium, sodium.

#153. A method according to item 152, where the assessment of anychemical constituent is based on spectrophotometric measurement, thespectrophotometric measurement being one or several of, but not limitedto; mid-infrared attenuation, near-infrared attenuation, visibleattenuation, ultra-violet attenuation, photoluminescence, raman scatter,nuclear magnetic resonance.

#154. A method according to item 152 through 153, where the assessmentof any chemical constituent is based on potentiometric measurement,preferably by the use of ion selective electrode.

#155. A method according to any of the items 142 through 154, where thesample material is either a milk sample used for heard improvementpurposes, or a milk sample used in a payment scheme.

#156. A method according to any of the items 142 through 155, where thesample material is a milk sample taken from one quarter of the udder,preferably where the purpose of the assessment of biological particlesis to determine the status of health.

#157. A method according to any of the items 1 through 132 or item 136,where the assessment is the determination of the number of somatic cellsin a volume of blood or a blood product, the type of the blood or bloodproduct being one or several of the following: human blood, animalblood, cow blood, goats blood, sheep blood, or buffalo blood.

#158. A method according to any of the items 1 through 132 or item 135,where the assessment is the determination of the number of bacteria in avolume of blood or a blood product, the type of the blood or bloodproduct being one or several of the following: human blood, animalblood, cow blood, goats blood, sheep blood, or buffalo blood.

#159. A method according to any of the items 1 through 132 or item 135,where the assessment is the determination of the types of bacteria in avolume of blood or a blood product, the type of the blood or bloodproduct being one or several of the following: human blood, animalblood, cow blood, goats blood, sheep blood, or buffalo blood.

#160. A method according to any of the items 1 through 132 or any ofitem 135 or 136, where the assessment is the estimation of rate ofsedimentation of biological particles in a volume of blood or a bloodproduct, the type of the blood or blood product being one or several ofthe following: human blood, animal blood, cow blood, goats blood, sheepblood, or buffalo blood.

#161. A method according to any of the items 1 through 132 or item 138,where the assessment is the determination of the number of somatic cellsin a volume of urine or a urine product, the type of the urine or urineproduct being one or several of the following: human urine, animalurine, cow urine, goats urine, sheep urine, or buffalo urine.

#162. A method according to any of the items 1 through 132 or item 137,where the assessment is the determination of the number of bacteria in avolume of urine or a urine product, the type of the urine or urineproduct being one or several of the following: human urine, animalurine, cow urine, goats urine, sheep urine, or buffalo urine.

#163. A method according to any of the items 1 through 132 or item 137,where the assessment is the determination of the types of bacteria in avolume of urine or a urine product, the type of the urine or urineproduct being one or several of the following: human urine, animalurine, cow urine, goats urine, sheep urine, or buffalo urine.

#164. A method according to any of the items 1 through 132 or any ofitem 137 or 138, where the assessment is the estimation of rate ofsedimentation of biological particles in a volume of urine or a urineproduct, the type of the urine or urine product being one or several ofthe following: human urine, animal urine, cow urine, goats urine, sheepurine, or buffalo urine.

#165. A method according to any of the items 157 through 164, where thepurpose of the assessment is to obtain information about the status ofhealth, such as infection, preferably where the assessment is carriedout in medical doctor office, physician office or veterinary office.

#166. A method according to any of the items 157 through 165, where theassessment is carried out substantially simultaneously with theassessment of the amount of any constituent in said sample material,preferably by using substantially a same portion of the sample materialfor the assessment, said constituent being one or several of, but notlimited to: fat, cholesterol, protein, lactose, urea, citric acid,glucose, ketones, carbon dioxide, oxygen, pH, potassium, calcium,sodium.

#167. A method according to any of the items 157 through 166, where saidassessment of any chemical constituent is based on spectrophotometricmeasurement, the spectrophotometric measurement being one or several of,but not limited to; mid-infrared attenuation, near-infrared attenuation,visible attenuation, ultra-violet attenuation, photoluminescence, ramanscatter, nuclear magnetic resonance.

#168. A method according to any of the items 157 through 167, where saidassessment of any chemical constituent is based on potentiometricmeasurement, preferably by the use of ion selective electrode.

#169. A method according to any of the preceding items, wheresubstantially entirely all the sample material used for the assessmentalong with any components intentionally added to the sample material orportion of the sample material is returned to a vial after thecompletion of the assessment, preferably the vial being substantiallyclosed to prevent spilling or evaporation of any material containedwithin the vial, more preferably the vial prior to the addition of anysample material, contains one or more chemical components, the functionof the chemical components being one, or several, but not limited to:substantial inhibitation of bacterial growth, substantial inhibitationof growth of fungus.

#170. A method according to any of the preceding items, where the samplematerial to be measured is contained in substantially closed, preferablywhere the container, or at least a part of the container, can be used asa sample compartment, substantially entirely all the sample materialused for the assessment along with any components intentionally added tothe sample material or portion of the sample material is retained in thecontainer after the completion of the measurement.

Sample Media

#171. A method according to any of the preceding items, where the samplebeing analysed is substantially an aqueous solution or an organicsolution.

#172. A method according to any of the preceding items, where the samplebeing analysed contains two or more phases in suspension, at least oneof the phases being inmiscible under the condition the measurements arecarried out.

#173. A method according to any of the preceding items, where all thephases of the sample are substantially liquid under the condition themeasurements are carried out.

#174. A method according to any of the preceding items, where at leastone of the phases of the sample is/are substantially solid under thecondition the measurements are carried out.

#175. A method according to any of the preceding items, where the samplecontains material, the material being dissolved and/or suspended, theamount of the material being substantially more than or equal 25%,preferably less than 25%, more preferably less than 10%, more preferablyless than 5%, more preferably less than 1%, more preferably less than0.1%, more preferably less than 0.01%, more preferably less than 0.001%,more preferably less than 0.0001%, more preferably less than 0.00001%,more preferably less than 0.000001%, of the total weight of said sample.

#176. A method according to any of the preceding items, wheresubstantially entirely no components have intentionally been added tothe sample being analysed.

#177. A method according to any of the preceding items, where the samplehas been intentionally modified by the addition of 1 solid, liquid,dissolved or suspended component equivalent to more than or equal to50%, preferably less than 50%, more preferably less than 35, morepreferably less than 20%, more preferably less than 10%, more preferablyless than 5%, more preferably less than 2%, more preferably less than1%, more preferably less than 0.1%, more preferably less than 0.01%,more preferably less than 0.001%, more preferably less than 0.0001%,more preferably less than 0.00001%, more preferably less than 0.000001%of the total weight of the sample.

#178. A method according to item 177, where the addition comprises morethan or equal to 10, preferably less than 10, more preferably less than6, more preferably less than 4, more preferably less than 3 solid,liquid, dissolved, or suspended components.

#179. A method according to item 177 or 178, where said addition of said1 or more components enhances the signal detected from the objects in asample.

#180. A method according to any of the items 177 through 179, where saidaddition of said 1 or more components suppresses one or more signalsfrom the sample which is being measured, the signal being a signalinterfering with the signal detected from the biological particles in asample.

#181. A method according to any of the items 177 through 179, where oneof intentionally added chemical components has the effect of adjustingthe pH of the sample before or during the assessment, the chemicalcomponent being one or several of the following, but not limited to:citric acid, citrate, acidic acid, acetate, phosphor acid, phosphate,carbonate, bicarbonate, boric acid, borate.

#182. A method according to any of the items 177 through 179, where oneintentionally added chemical components has the effect of adjusting thepH of the sample before or during the assessment, the chemical componentbeing a mixture of citric acid and citrate.

#183. A method according to any of the items 177 through 179, where oneintentionally added chemical components has the effect of enhancing anysignal detected from the biological particles, the chemical componentbeing a mixture of citric acid and citrate.

#184. A method according to any of the items 177 through 179, where oneof intentionally added chemical components has the effect of surfactant,the chemical component being of one or several of the following groupsof surfactants, but not limited to: anionic surfactant, cationicsurfactant, amphoteric surfactant, nonionic surfactant.

#185. A method according to item 184 where one intentionally addedchemical component is t-Octylphenoxypolyethoxyethanol (Triton X-100).

#186. A method according to any of the items 177 through 179, where oneof intentionally added chemical components has the effect of binding oneor several of metal ions present in the sample, preferably the chemicalcomponent being one capable of forming a metal ion complex with themetal ion.

#187. A method according to item 186, where the intentionally addedchemical component is one or several of the following, but not limitedto: EDTA, Oxalic acid, Oxalate, Ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetec acid (EGTA).

Type of Objects

#188. A method according to any of the preceding items, where thebiological particles to be assessed are one or several of the following,but not limited to: somatic cells, red blood cells, blood platelets,bacteria, yeast cells, fragments of cells, lipid globules, proteinmicelles, plankton, algae.

#189. A method according to any of the preceding items, where thebiological particles to be assessed comprises polymer beads bound tobiological particles or components in connection with assessment ofthese biological molecules or components.

Type of Specimen

#190. A method according to any of the preceding items, where the samplematerial is one or several of the following, but not limited to:specimen of human origin, specimen of animal origin, drinking water,waste water, process water, sea water, lake water, river water, groundwater, water used for heating, water used for cooling water used foradjusting humidity, water used for washing or bathing, water used inpool or swimming pool, food, feed or components of food and feed, milkor a milk product, blood or a blood product, urine, faeces, saliva,specimen from an inflammation, specimen from the petrochemical industry,specimen from the pharmaceutical industry, specimen from the food orfeed industry.

Object Size

# 191. A method according to any of the preceding items, where theaverage size of the biological particle to be assessed is less than 0.01μm, preferably less than 0.1 μm, more preferably less than 1 μm, morepreferably less than 2 μm, more preferably less than 3 μm, morepreferably less than 4 μm, more preferably less than 6 μm, morepreferably less than 10 μm, more preferably less than 20 μm, morepreferably less than 50 μm, more preferably less than 100 μm.

#192. A method according to any of the preceding items, where theaverage size of the biological particle to be assessed is larger than orequal to 100 μm, preferably larger than 150 μm, more preferably largerthan 200 μm, more preferably larger than 400 μm.

Licences

Somatic Cells in Milk

#193. A method according to any of the preceding items, wherein thebiological particles are somatic cells or fragments thereof, and thesample material is a milk sample, the sample of the sample material isilluminated in the sample compartment with electromagnetic radiationwhere at least a portion of said electromagnetic radiation has energywhich can give rise to a photoluminescence signal, preferablyfluorescent signal, the signal originating at least from said somaticcells or portions of said somatic cells or components interacting withor bound to the somatic cells or portions thereof.

#194. A method according to item 193, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the somatic cells or parts ofthe somatic cells, preferably by binding to or interacting with DNAmaterial contained within or originating from the somatic cells.

On-Farm

#195. A method according to any of items #1 through #192, wherein thebiological particles are somatic cells or fragments thereof, and thesample material is a milk sample, the purpose of the assessment being toobtain information about the health status of a milking animal,preferably to obtain information about subclinical or clinical mastitis,the sample of the sample material is placed in a sample compartment bythe use of a flow means capable of replacing the sample within thesample compartment with a different sample, the sample of the samplematerial is illuminated in the sample compartment with electromagneticradiation where at least a portion of said electromagnetic radiation hasenergy which can give rise to a photoluminescence signal, preferablyfluorescent signal, the signal originating at least from said somaticcells or portions of said somatic cells or components interacting withor bound to the somatic cells or portions thereof.

#196. A method according to item 195, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the somatic cells or parts ofthe somatic cells, preferably by binding to or interacting with DNAmaterial contained within or originating from the somatic cells.

Central Laboratory

#197. A method according to any of items #1 through #192, wherein thebiological particles are somatic cells or fragments thereof, and thesample material is a milk sample, the sample of the sample material isplaced in a sample compartment by the use of a flow means capable ofreplacing the sample within the sample compartment with a differentsample the time between the replacement of sample material being shorterthan 30 seconds, preferably shorter than 15 seconds, more preferablyshorter than 10 seconds, the sample of the sample material isilluminated in the sample compartment with electromagnetic radiationwhere at least a portion of said electromagnetic radiation has energywhich can give rise to a photoluminescence signal, preferablyfluorescent signal, the signal originating at least from said somaticcells or portions of said somatic cells or components interacting withor bound to the somatic cells or portions thereof.

#198. A method according to item 197, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the somatic cells or parts ofthe somatic cells, preferably by binding to or interacting with DNAmaterial contained within or originating from the somatic cells.

On-Line

#199. A method according to any of items #1 through #192, wherein thebiological particles are somatic cells or fragments thereof, and thesample material is a milk sample, the assessment being performedsubstantially at the beginning of milking, or during milking, orimmediately after milking has taken place, the sample of the samplematerial is placed in a sample compartment by the use of a flow meanscapable of replacing the sample within the sample compartment with adifferent sample flowing milk directly from a milking unit or flowingmilk from an intermediate reservoir which is gradually filled duringmilking, preferably where said reservoir is filled with milksubstantially representing the composition of the total volume of milkbeing milked, the sample of the sample material is illuminated in thesample compartment with electromagnetic radiation where at least aportion of said electromagnetic radiation has energy which can give riseto a photoluminescence signal, preferably fluorescent signal, the signaloriginating at least from said somatic cells or portions of said somaticcells or components interacting with or bound to the somatic cells orportions thereof.

#200. A method according to item 199, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the somatic cells or parts ofthe somatic cells, preferably by binding to or interacting with DNAmaterial contained within or originating from the somatic cells.

Disposable Cuvette

#201. A method according to any of items #1 through #192, wherein thebiological particles are somatic cells or fragments thereof, and thesample material is a milk sample, a portion of the sample material isplaced in a sample compartment being at least a part of a unit which canbe replaced between substantially every assessment or where each of saidunits can only be used for said assessment of one of said samplematerials, the sample of the sample material is illuminated in thesample compartment with electromagnetic radiation where at least aportion of said electromagnetic radiation has energy which can give riseto a photoluminescence signal, preferably fluorescent signal, the signaloriginating at least from said somatic cells or portions of said somaticcells or components interacting with or bound to the somatic cells orportions thereof.

#202. A method according to item 201, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the somatic cells or parts ofthe somatic cells, preferably by binding to or interacting with DNAmaterial contained within or originating from the somatic cells.

Bacteria in Milk

#203. A method according to any of items #1 through #192, wherein thebiological particles are bacteria or fragments thereof, and the samplematerial is a milk sample, the sample of the sample material isilluminated in the sample compartment with electromagnetic radiationwhere at least a portion of said electromagnetic radiation has energywhich can give rise to a photoluminescence signal, preferablyfluorescent signal, the signal originating at least from said bacteriaor portions of said bacteria or components interacting with or bound tothe bacteria or portions thereof.

#204. A method according to item 203, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the bacteria or parts of thebacteria, preferably by binding to or interacting with DNA materialcontained within or originating from the bacteria.

On-Farm

#205. A method according to any of items #1 through #192, wherein thebiological particles are bacteria or fragments thereof, and the samplematerial is a milk sample, the purpose of the assessment being to obtaininformation about the health status of a milking animal, preferably toobtain information about subclinical or clinical mastitis, the sample ofthe sample material is placed in a sample compartment by the use of aflow means capable of replacing the sample within the sample compartmentwith a different sample, the sample of the sample material isilluminated in the sample compartment with electromagnetic radiationwhere at least a portion of said electromagnetic radiation has energywhich can give rise to a photoluminescence signal, preferablyfluorescent signal, the signal originating at least from said bacteriaor portions of said bacteria or components interacting with or bound tothe bacteria or portions thereof.

#206. A method according to item 205, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the bacteria or parts of thebacteria, preferably by binding to or interacting with DNA materialcontained within or originating from the bacteria.

Central Laboratory

#207. A method according to any of items #1 through #192, wherein thebiological particles are bacteria or fragments thereof, and the samplematerial is a milk sample, the sample of the sample material is placedin a sample compartment by the use of a flow means capable of replacingthe sample within the sample compartment with a different sample thetime between the replacement of sample material being shorter than 30seconds, preferably shorter than 15 seconds, more preferably shorterthan 10 seconds, the sample of the sample material is illuminated in thesample compartment with electromagnetic radiation where at least aportion of said electromagnetic radiation has energy which can give riseto a photoluminescence signal, preferably fluorescent signal, the signaloriginating at least from said bacteria or portions of said bacteria orcomponents interacting with or bound to the bacteria or portionsthereof.

#208. A method according to item 207, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the bacteria or parts of thebacteria, preferably by binding to or interacting with DNA materialcontained within or originating from the bacteria.

On-Line

#209. A method according to any of items #1 through #192, wherein thebiological particles are bacteria or fragments thereof, and the samplematerial is a milk sample, the assessment being performed substantiallyat the beginning of milking, or during milking, or immediately aftermilking has taken place, the sample of the sample material is placed ina sample compartment by the use of a flow means capable of replacing thesample within the sample compartment with a different sample flowingmilk directly from a milking unit or flowing milk from an intermediatereservoir which is gradually filled during milking, preferably wheresaid reservoir is filled with milk substantially representing thecomposition of the total volume of milk being milked, the sample of thesample material is illuminated in the sample compartment withelectromagnetic radiation where at least a portion of saidelectromagnetic radiation has energy which can give rise to aphotoluminescence signal, preferably fluorescent signal, the signaloriginating at least from said bacteria or portions of said bacteria orcomponents interacting with or bound to the bacteria or portionsthereof.

#210. A method according to item 209, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the bacteria or parts of thebacteria, preferably by binding to or interacting with DNA materialcontained within or originating from the bacteria.

Disposable Cuvette

#211. A method according to any of items #1 through # 192, wherein thebiological particles are bacteria or fragments thereof, and the samplematerial is a milk sample, a portion of the sample material is placed ina sample compartment being at least a part of a unit which can bereplaced between substantially every assessment or where each of saidunits can only be used for said assessment of one of said samplematerials, the sample of the sample material is illuminated in thesample compartment with electromagnetic radiation where at least aportion of said electromagnetic radiation has energy which can give riseto a photoluminescence signal, preferably fluorescent signal, the signaloriginating at least from said bacteria or portions of said bacteria orcomponents interacting with or bound to the bacteria or portionsthereof.

#212. A method according to item 211, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the bacteria or parts of thebacteria, preferably by binding to or interacting with DNA materialcontained within or originating from the bacteria.

Somatic cells in Blood

#213. A method according to any of items #1 through #192, wherein thebiological particles are somatic cells or fragments thereof, and thesample material is a blood sample, the sample of the sample material isilluminated in the sample compartment with electromagnetic radiationwhere at least a portion of said electromagnetic radiation has energywhich can give rise to a photoluminescence signal, preferablyfluorescent signal, the signal originating at least from said somaticcells or portions of said somatic cells or components interacting withor bound to the somatic cells or portions thereof.

#214. A method according to item 213, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the somatic cells or parts ofthe somatic cells, preferably by binding to or interacting with DNAmaterial contained within or originating from the somatic cells.

Disposable Cuvette

#215. A method according to any of items # 1 through # 192, wherein thebiological particles are somatic cells or fragments thereof, and thesample material is a blood sample, a portion of the sample material isplaced in a sample compartment being at least a part of a unit which canbe replaced between substantially every assessment or where each of saidunits can only be used for said assessment of one of said samplematerials, the sample of the sample material is illuminated in thesample compartment with electromagnetic radiation where at least aportion of said electromagnetic radiation has energy which can give riseto a photoluminescence signal, preferably fluorescent signal, the signaloriginating at least from said somatic cells or portions of said somaticcells or components interacting with or bound to the somatic cells orportions thereof.

#216. A method according to item 215, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the somatic cells or parts ofthe somatic cells, preferably by binding to or interacting with DNAmaterial contained within or originating from the somatic cells.

Somatic cells in Urine

#217. A method according to any of items #1 through #192, wherein thebiological particles are somatic cells or fragments thereof, and thesample material is a urine sample, a portion of the sample material isplaced in a sample compartment being at least a part of a unit which canbe replaced between substantially every assessment or where each of saidunits can only be used for said assessment of one of said samplematerials, the sample of the sample material is illuminated in thesample compartment with electromagnetic radiation where at least aportion of said electromagnetic radiation has energy which can give riseto a photoluminescence signal, preferably fluorescent signal, the signaloriginating at least from said somatic cells or portions of said somaticcells or components interacting with or bound to the somatic cells orportions thereof.

#218. A method according to item 217, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the somatic cells or parts ofthe somatic cells, preferably by binding to or interacting with DNAmaterial contained within or originating from the somatic cells.

Disposable Cuvette

#219. A method according to any of items #1 through #192, wherein thebiological particles are somatic cells or fragments thereof, and thesample material is a urine sample, a portion of the sample material isplaced in a sample compartment being at least a part of a unit which canbe replaced between substantially every assessment or where each of saidunits can only be used for said assessment of one of said samplematerials, the sample of the sample material is illuminated in thesample compartment with electromagnetic radiation where at least aportion of said electromagnetic radiation has energy which can give riseto a photoluminescence signal, preferably fluorescent signal, the signaloriginating at least from said somatic cells or portions of said somaticcells or components interacting with or bound to the somatic cells orportions thereof.

#220. A method according to item 219, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the somatic cells or parts ofthe somatic cells, preferably by binding to or interacting with DNAmaterial contained within or originating from the somatic cells.

Bacteria in Urine

#221. A method according to any of items #1 through #192, wherein thebiological particles are bacteria or fragments thereof, and the samplematerial is a urine sample, a portion of the sample material is placedin a sample compartment being at least a part of a unit which can bereplaced between substantially every assessment or where each of saidunits can only be used for said assessment of one of said samplematerials, the sample of the sample material is illuminated in thesample compartment with electromagnetic radiation where at least aportion of said electromagnetic radiation has energy which can give riseto a photoluminescence signal, preferably fluorescent signal, the signaloriginating at least from said bacteria or portions of said bacteria orcomponents interacting with or bound to the bacteria or portionsthereof.

#222. A method according to item 221, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the bacteria or parts of thebacteria, preferably by binding to or interacting with DNA materialcontained within or originating from the bacteria.

Disposable Cuvette

#223. A method according to any of items #1 through #192, wherein thebiological particles are bacteria or fragments thereof, and the samplematerial is a urine sample, a portion of the sample material is placedin a sample compartment being at least a part of a unit which can bereplaced between substantially every assessment or where each of saidunits can only be used for said assessment of one of said samplematerials, the sample of the sample material is illuminated in thesample compartment with electromagnetic radiation where at least aportion of said electromagnetic radiation has energy which can give riseto a photoluminescence signal, preferably fluorescent signal, the signaloriginating at least from said bacteria or portions of said bacteria orcomponents interacting with or bound to the bacteria or portionsthereof.

#224. A method according to item 223, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the bacteria or parts of thebacteria, preferably by binding to or interacting with DNA materialcontained within or originating from the bacteria.

Bacteria Water

#225. A method according to any of items #1 through #192, wherein thebiological particles are bacteria or fragments thereof, and the samplematerial is a water sample, a portion of the sample material is placedin a sample compartment being at least a part of a unit which can bereplaced between substantially every assessment or where each of saidunits can only be used for said assessment of one of said samplematerials, the sample of the sample material is illuminated in thesample compartment with electromagnetic radiation where at least aportion of said electromagnetic radiation has energy which can give riseto a photoluminescence signal, preferably fluorescent signal, the signaloriginating at least from said bacteria or portions of said bacteria orcomponents interacting with or bound to the bacteria or portionsthereof.

#226. A method according to item 225, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the bacteria or parts of thebacteria, preferably by binding to or interacting with DNA materialcontained within or originating from the bacteria.

Disposable Cuvette

#227. A method according to any of items #1 through # 192, wherein thebiological particles are bacteria or fragments thereof, and the samplematerial is a water sample, a portion of the sample material is placedin a sample compartment being at least a part of a unit which can bereplaced between substantially every assessment or where each of saidunits can only be used for said assessment of one of said samplematerials, the sample of the sample material is illuminated in thesample compartment with electromagnetic radiation where at least aportion of said electromagnetic radiation has energy which can give riseto a photoluminescence signal, preferably fluorescent signal, the signaloriginating at least from said bacteria or portions of said bacteria orcomponents interacting with or bound to the bacteria or portionsthereof.

#228. A method according to item 227, wherein the signal originates fromone or several types of molecules intentionally added to said samplewhich interact or bind to or interact with the bacteria or parts of thebacteria, preferably by binding to or interacting with DNA materialcontained within or originating from the bacteria.

#229. A device capable of the assessment of biological particles in avolume of liquid sample material according to any of the precedingitems.

#229. A device capable of the assessment of biological particles in avolume of liquid sample material according to any of the items 1 through229 comprising at least means for flowing the sample to a samplecompartment, means for the detection of any signal from the sample andmeans for the electronical or digital transformation of any such signal.

#229. A device capable of the assessment of biological particles in avolume of liquid sample material according to any of the items 1 through229 comprising at least a sample compartment, means for the detection ofany signal from the sample and means for the electronical or digitaltransformation of any such signal.

Multiple Exposure

In order to allow optimal assessment of particles it is possible to basesuch assessment on a number of measurements which are taken from asample. One advantage is to make repeated measurement of the sameportion of a sample, thus improving any signal to noise condition by theuse of propagation of error. Another aspect is to increase the totalvolume of sample which is analysed by taking more than one measurementsfrom different portion of a sample.

Under some conditions it can be advantageous to adjust the measurementtime, for instance when the intensity of signals is varying, maybedepending on which type of particles are being analysed.

Detection Error

The present invention offers methods for the assessment of the number ofbiological particles in a sample with total error, expressed as relativeprediction error in percent of the average number of particles in avolume, which preferably is less than 30% and often as low as 10% oreven as low or less than 1%. This can be obtained for instance bycontrolling the volume of sample which is analysed.

Sample Throughput

The method of the present invention allow the assessment of biologicalparticles at a rate which preferably amounts to 10 or more assessmentsper hour, and even as many as 100 or more assessments per hour, even asmany as 1000 or more assessments per hour.

It is also possible to combine more than one more or less identicalanalysing systems, in such a way that they work in parallel and therebyconstitute a method which can assess even higher number of samples perhour.

Detection Limits

The extensive flexibility of the method of the present invention, makesit possible to analyse volumes, which can allow the assessment ofparticles in samples where the total number of such particles per volumeis ranging from more than 1×10⁸ particles per ml sample to less and 1particle per ml sample. one of the most important aspect for thatpurpose being the total volume being analysed.

Signal Source

The signals which the assessment of particles can be based on arevirtually any type of electromagnetic radiation, and in particular wherethe source of such electromagnetic radiation or mechanism havinginfluence of it can be photoluminescence with lifetime of the exitedstate of less than or equal to 10⁻⁶ seconds, photoluminescence withlifetime of the exited state of garter than 10⁻⁶ seconds,chemiluminescence, rayleigh scatter, raman scatter, attenuation ofelectromagnetic radiation, absorption of the electromagnetic radiation,scatter of the electromagnetic radiation.

Wavelength Sensitivity

When the signal being detected is an electromagnetic radiation it ispreferred to use a detection element which is sensitive to suchradiation. Preferred embodiments use arrays of detection elements whichare sensitive to electromagnetic radiation of wavelength in one orseveral of the following regions: 100 nm to 200 nm 200 nm to 600 nm, 300nm to 700 nm, 400 nm to 800 nm, 600 nm to 1 μm, 800 nm to 2 μm, 2 μm to10 μm, 5 μm to 10 μm, 10 μm to 20 μm, 20 μm to 40 μm.

Wavelength Separation

It is often of interest to separate electromagnetic radiation intoseparate wavelengths or wavebands, especially when the source of suchradiation emits energy over a broad spectrum of wavelengths. In methodsof assessment of particles which are based on attenuation of energy,detection of emitted or scattered energy or the like, the ability ofbeing able to separate energy into separate wavelengths or wavebands, isimportant. This applies also to any electromagnetic radiation whichoriginates from within the sample, for instance by means ofphotoluminescence or chemiluminescence. In some methods of thisinvention it is possible to use information obtained when using two ormore different wavelengths or wavebands, for instance to distinguishbetween two or more types of particles on the bases of how those reactto different energies.

One method of performing such wavelength separation is to illuminate aportion of a sample with more than one wavelength or wavebandsimultaneously, preferably in such a way that different portions of thesample are illuminated with different wavelengths or wavebands ofenergy. This is particularly of interest when the assessment ofparticles concerns the identification of one or more types of particlessince particles are exposed to different wavelength energy depending ontheir position within the sample compartment.

Another method of performing such wavelength separation is to separateany electromagnetic radiation emitted from the sample, preferably wheremore than two detection elements observe signals from substantially thesame portion of the sample but due to the wavelength separation thesedetection elements detect different wavelengths or wavebands of energy.Thereby it is possible to derive spectral characteristics of anyparticle which can be used for its assessment.

One method is the intensity modulation of electromagnetic radiation.When such modulation is controlled it is possible to use it for theimprovement of signal to noise ratio, for instance by observing anybackground signal in a period where the intensity is low or zero, andthen correcting any signal measured when the intensity is high, by thebackground information.

It is also possible to frequency modulate electromagnetic radiation bythe use of an optically active crystals or by the use of aninterferometer. The effect of such modulation could be to obtainspectral information about any particle present in the sample.

Light Sources

In methods based on attenuation of electromagnetic radiation, orillumination of the sample it is preferred to use a source of radiationsuch as light emitting diodes, lasers, laser diodes, thermal lightsource or gas discharge lamp. When the intensity of the illuminatingenergy is of interest it is possible to use more than one energy source,even when the different light sources have different energy spectrum,for instance when two different light emitting diodes are used whichemit energy in different wavebands.

To improve the efficiency of such light source in illuminating thesample it is often desirable to use a focusing system for focusingenergy onto the sample.

Reflection

When the electromagnetic radiation is used to illuminate a sample forthe purpose of causing photoluminescence or the like, it is of interestto be able to increase the efficiency of such radiation source by beingable to reflect any energy which is transmitted through the sample backonto the sample, preferably by the use of a reflecting means, forinstance dichroic mirrors, which reflect energy of certain wavelengthswhile allowing the transmission of energies at other wavelengths.

Incidence Angle.

In a method of this invention using photoluminescence as sources ofdetected signal, it is possible to arrange the light source, relative tothe axis of the sample compartment, and particularly relative to an axiswhich the array of detection elements and the sample compartment form,in such a way that the angle between the axis and the light source isbetween 0 and 180 degrees.

Detector Types

As detection elements, it is possible to use one of several commerciallyavailable arrays of detection elements, such as array of charge coupleddevices (CCD) or array of light sensitive diodes (CMOS image sensor).Such arrays of detection elements can have on-chip integrated signalcondition and/or signal processing facilities.

Signal Conditioning—Software

At least one embodiment makes use of a calculating means, such as adigital computer, which at least can be used for the correction of anymeasured signal for a systematic or varying bias, for instance by usingone or more predetermined variable to adjust the measured signal. Thedetermination of the predefined variable can be done on the bases ofvalues form measured signals of one or more reference element situatedclose to the element being corrected, or it can be done on the bases ofvalues from one or several of any other measurements.

In particular it is of interest to subtract one of the other measuredsignals, often one of previously measured signals, from the measuredsignal, thereby removing any bias effect which also was present in theother measurement. The other measurement can be another measurement of adifferent portion of the same sample, or a measurement of a differentsample.

Such calculating means can also be used to correct a measured signal forvariation in sensitivity, by using one or more predefined variable. Thedetermination of the predefined variable can be done on the bases ofvalues form measured signals of one or more reference element situatedclose to the element being corrected, or it can be done on the bases ofvalues from one or several of any previous measurements.

One suitable method for the correction of measured signals is tosubtract one result from an array of detection elements from anotherresults, obtained form a different portion of the same sample, or from adifferent sample. Such subtraction has the effect of reducing, orremoving any systematic variation in baseline levels of the signals fromthe array of detection elements, caused by dark-current or possibly byparticles which are immobilised on the interior of the samplecompartment, any assessment being based on substantially only thepositive results of such subtraction. If the two measurement originatefrom different portion of the same sample, then it is also possible toperform assessment on the bases of the result from the subtraction bytreating any negative result from the subtraction as positive number,thus effectively performing an assessment of more than one measurementwith the same efforts as when a single measurement is used. In this wayit is possible to combine the result from more than one subtraction,preferably as many as the actual noise level allows, for instance bykeeping the noise in the combined results less than a given fraction ofthe smallest signals which are used for the assessment of particles.

Image Processing

The present invention is well suited for making use of state of the artimage processing in the assessment of particles, for instance when theassessment is the identification of one or more of different types ofparticles.

Power

Any instrument constructed according to the present invention can beoperated on electrical power, such as 110 or 220 V AC, by the use ofappropriate transformer system. A battery or an accumulator can also beused as a source of power, and this is in particular of interest whenthe instrument is intended for use where the transport of the instrumentis required. Such battery or accumulator can also be one which can berecharged, thereby making it possible to regenerate and reuse.

1. A method for the assessment of at least one quantity parameter and/orat least one quality parameter of biological particles in a liquidanalyte material, comprising providing a volume of between 0.01 μl and20 μl of a liquid sample comprising the liquid analyte material,exposing, onto an array of active detection elements, an at leastone-dimensional spatial image of said volume, the image being one whichis detectable as an intensity by individual active detection elements,under conditions which will permit processing of the intensitiesdetected by the array of detection elements during the exposure in sucha manner that images of electromagnetic signals from the biologicalparticles are identified as distinct from images of electromagneticsignals from background signals, and wherein the spatial image exposedonto the array of active detection elements is subject to such a linearenlargement that the ratio of the image of a linear dimension on thearray of detection elements to the original linear dimension in theexposing domain is smaller than 10:1, the sample being at stand stillduring the exposure, processing the intensities detected by thedetection elements in such a manner that signals from the biologicalparticles are identified as distinct from background signals, andcorrelating the results of the processing to the at least one quantityparameter and/or the at least one quality parameter of the liquidanalyte material.
 2. The method according the claim 1, wherein thevolume is provided in a sample compartment having a wall part definingan exposing area, the wall part allowing electromagnetic from the samplein the compartment to pass through the wall and to be exposed to theexterior.
 3. A method according to claim 1, wherein at least a majorpart of the electromagnetic radiation emitted from the sample duringexposure originates from or is caused by electromagnetic radiationsupplied to the sample from a light source, at least a major part of theradiation from the light source having a direction which is transverseto the wall of the sample compartment or a plane defined by the domain.4. A method according to claim 1, wherein the spatial image of thevolume is a two-dimensional image.
 5. A method according to claim 1,wherein the array of detection elements is arranged in such a way thatthe a series of detection elements from a substantially straight line.6. A method according to claim 5, wherein the array of detectionelements is arranged in two directions in such a way that the detectionelements form a series of substantially parallel straight lines, theseries forming a rectangle.
 7. A method according to claim 1, whereinthe exposure of the spatial image of volume onto the array of detectionelements is performed by focusing an image of electromagnetic signalsfrom at least a part of the exposing domain onto the array of detectionelements by means of a focusing means.
 8. A method according to claim 1,wherein the ratio is at the most 6:1.
 9. A method according to claim 1,wherein the ratio is in the range between 10:1 and 1:10.
 10. A methodaccording to claim 8, wherein the particles the parameter or parametersof which is/are to be assessed are of a size between 3 μm and 100 μm andthe ratio is in the range between 3:1 and 1:100.
 11. A method accordingto claim 1, wherein the individual particles the parameter or parametersof which is/are to be assessed are imaged on at most 25 detectionelements.
 12. A method according to claim 1, wherein the interior of thesample compartment has an average thickness of between 20 μm and 2000μm.
 13. A method according to claim 1, wherein the sample compartmenthas dimensions, in a direction substantially parallel to the array ofdetection elements, in the range between 1 mm by 1 mm and 10 mm by 10mm.
 14. A method according to claim 1, wherein the particles theparameter or parameters of which is/are to be assessed are of a size ofbetween ⅓ μm to 3 μm, and the volume of the liquid sample from whichelectromagnetic radiation is exposed onto the array is in the rangebetween 0.01 μl and 1 μl.
 15. A method according to claim 1, wherein theparticles the parameter or parameters of which is/are to be assessed areof a size between 3 μm to 100 μm, ant the volume of the liquid samplefrom which electromagnetic radiation is exposed onto the array is in therange between 0.04 μl and 4 μl.
 16. A method according to claim 1,wherein the parameter to be assessed is the number of the biologicalparticles per volume of the liquid analyte material.
 17. A methodaccording to claim 1, wherein the parameter(s) so be assessed is thesize and/or shape of the biological particles in the liquid analytematerial.
 18. A method according to claim 1, wherein the signal which isdetected by the detecting elements originates from one or several typesof molecules of types which bind to, are retained within, or interactwith, the biological particles, such molecules being added to the sampleor the isolated particles before or during exposure, the molecules beingmolecules giving rise to one or several of the following phenomena:attenuation of electromagnetic radiation, photoluminescence whenilluminated with electromagnetic radiation, scatter of electromagneticradiation, raman scatter.
 19. A method according to claim 1, wherein thesignal which is detected by the detecting elements originates from oneor several types of molecules of types which bind to, are retainedwithin, or interact with, the biological particles, such molecules beingadded to the sample to the isolated particles, the molecules causing, orenhancing the emission of electromagnetic radiation such aschemiluminescence or photoluminescence, such as fluorescence orphotoluminescence, such as fluorescence or phosphorescence, when excitedwith radiation which is substantially higher in energy than the emittedphotoluminescence.
 20. A method according to claim 1, wherein theduration of the exposure is in the range from 100 milliseconds to 5seconds.
 21. A method according to claim 20, wherein the exposure isperformed as a single exposure.
 22. A method according to claim 1,wherein compression of information of the intensities representingdistinct objects scattered over an area, and object being represented bya variation in the intensity information said information existing inthe form of varying degrees of measurable intensity of a physicalproperty distributed over a confined area divided into sub-areas, eachof which sub-areas having assigned thereto an index uniquely identifyingthe sub-area, the method comprising determining the intensity of thephysical property, a) defining a sub-area of interest situated in agroup of sub-areas comprising of at least 2×2 sub-areas situatedadjacent to each other, b) evaluating in said sub-area of interest atleast one directional derivative(s) of the measurable intensity in thesub-area of interest with respect to predetermined geometricaldirections(s) in the plane of the confined area, the directionalderivative(s) is (are) based on measurable intensities in sub-areassituated adjacent to or in proximity of the group of sub-area, c) basedon the evaluation of the at least one directional derivative anattribute is assigned to the value assigned to said sub-area ofinterest; the attribute represent an adjusted measurable intensityand/or information(s) related to a predetermined strategy for adjustmentof the measurable intensity in the sub-area of interest or sub-areassituated adjacent to or in proximity to the sub-area of interest, d)repeating the step a)-c) for substantially all sub-areas of the confinedarea.
 23. A method according to claim 1, wherein the correlationcomprises: identifying and counting substantially all detection elementshaving intensities which are distinct from background signals, adjustingthe result of the counting by a predefined scaling value, the scalingvalue being directly related to the number of detection elementsrepresenting a signal from a biological particle, the result of thescaling being correlated to the number of particles representedexposure.
 24. A method according to claim 23, where the measuredintensities of the detection elements have been adjusted prior tocounting, the adjustment comprising the steps of: a) defining a range ofa predetermined size in a co-ordinate system representing the intensityvalues of the detection elements, the size of the range being determinedsuch that it is bigger than the representation of an biological particlehaving an average extension, b) choosing a first detection element, thefirst detection element being one of which the intensity is subject toan adjustment, c) positioning the range such that the detection elementof which the intensity is to be adjusted is substantially in the centerof the range, d) adjusting the intensity of the detection element in thecenter of the range based on the result of an investigation of at leastone gradient describing the variation of the signal intensities insidethe range and around the center of the range by considering intensitiesof detection elements describing the gradient, and repeating the step b)through c) until a predetermined number of detection elements has beenadjusted a predetermined number of times.
 25. A method according toclaim 1, wherein the sample compartment is a removable samplecompartment, which can be readily removed from the measuring system. 26.A method according to claim 25, wherein the sample compartment comprisesone or more compartments, where chemical or physical components can bestored such that the chemical or physical components can be added to anysample present in the sample compartment one at a time or more than oneat a time.
 27. A method according to claim 1, wherein the identificationof a particle present in the sample is done by comparing the level ofsignal from each detection element with a predefined level, or to alevel which is estimated on the basis of neighboring detection elements.28. A system for the assessment of at least one quantity parameterand/or at least one quality parameter of biological particles in aliquid analyte material, comprising a sample compartment (104, 401, 501,606, 704, 807) defining a volume of between 0.01 μl and 20 μl of aliquid sample, an array of active detection elements (107, 404, 506,811), onto which an at lease one-dimensional spatial image of saidvolume is exposed, the image being one which is detectable as anintensity by individual active detection elements, under conditionswhich will permit processing of the intensities detected by the array ofdetection elements (107, 404, 506, 811) during the exposure in such amanner that images of electromagnetic signals from the biologicalparticles are identified as distinct from images of electromagneticsignals from background signals, a lens consisting of one or severalelements (105, 402, 403, 502, 503, 504, 505, 808) for focusing an imageof electromagnetic signals from at least part of the exposing domainonto the array of detection elements (107, 404, 506, 811) such that theratio of the image of a linear dimension on the array of detectionelements (107, 404, 506, 811) to the original linear dimension in theexposing domain is smaller than 10:1, the system being adapted to keepthe sample in the sample compartment (104, 401, 501, 606, 704, 807) atstand still during the exposure, a computer unit (813) for processingthe intensities detected by the detection elements in such a manner thatsignals from the biological particles are identified as distinct frombackground signals, and for correlating the results of the processing tothe at least one quantity parameter and/or the at least one qualityparameter of the liquid analyte material.
 29. A system according toclaim 28, wherein at least a major part of the electromagnetic radiationemitted from the sample during exposure originates from or is caused byelectromagnetic radiation supplied to the sample from a light source(101, 804), at least a major part of the radiation from the light sourcehaving a direction which is transverse to the wall of the samplecompartment (104, 807) or a plane defined by the domain.
 30. A systemaccording to claim 28, comprising of one or more valves (603, 607, 705)which can control the flow of sample or any other component.
 31. Asystem according to claim 28, wherein the array of detection elements(107, 404, 506, 811) is arranged in such a way that the a series ofdetection elements form a substantially straight line.
 32. A systemaccording to claim 31, wherein the array of detection elements (107,404, 506, 811) is arranged in two directions in such a way that thedetection elements form a series of substantially parallel straightlines, the series forming a rectangle.
 33. A system according to claim28, wherein the ratio is at the most 6:1.
 34. A system according toclaim 28, wherein the ratio is in the range between 10:1 and 1:10.
 35. Asystem according to claim 28, wherein the ratio is in the range between3:1 and 1:100.
 36. A system according to claim 28, wherein the interiorof the sample compartment (104, 401, 501, 606, 704, 807) has an averagethickness of between 20 μm and 2000 μm.
 37. A system according to claim28, wherein the sample compartment (104, 401, 501, 606, 704, 807) hasdimensions, in a direction substantially parallel to the array ofdetection elements, in the range between 1 mm by 1 mm and 10 mm by 10mm.
 38. The system according to claim 28, comprising a pump, said pumpbeing situated either upstream to the sample compartment (104, 401, 501,606, 704, 807) or downstream to the sample compartment (104, 401, 501,606, 704, 807).
 39. A system according to claim 28, comprising means toassess the number of the biological particles per volume of the liquidanalyte material.
 40. A system according to claim 28, comprising meansto assess the size and/or shape of the biological particles in theliquid analyte material.
 41. A system according to claim 28, wherein theduration of the exposure is in the range from 100 milliseconds to 5seconds.
 42. A system according to claim 28, where said array ofdetection elements is one or several of the following types: full frameCCD, frame transfer CCD, interline transfer CCD, line scan CCD.
 43. Asystem according to claim 28, where said array of detection elements isa CMOS image sensor, preferably a CMOS image sensor with on-chipintegrated signal condition and/or signal processing, more preferably aCMOS image sensor with on-chip integrated computing means capable ofperforming image processing.
 44. A system according to claim 28, wherethe transmission of electromagnetic radiation onto the sample isaccomplished by the use of illuminating means.
 45. A system according toclaim 44, where the electromagnetic radiation transmitted onto thesample is focused by a focusing mean (102), the focusing mean (102)having the effect of substantially increasing the intensity of saidelectromagnetic radiation in or at said sample.
 46. A system accordingto claim 28, wherein the identification of a particle present in thesample is done by comparing the level of signal from each detectionelement with a predefined level or by comparing to a level, which isestimated on the basis of the signals from neighboring detectionelements.
 47. A system according to claim 28, further comprising acontainer for one or more chemical components, the container beingconnected to a flow system where the sample flows, at least a portion ofthe sample flowing through the chemical container and thus allowing themixing of the chemical components with the sample.
 48. A systemaccording to claim 28, further comprising a source of excitation light(101, 804) and an appropriate set of optical filters (103, 106, 805,806, 809, 810).